Anti-dc-hil antibodies for cancer diagnosis, prognosis and therapy

ABSTRACT

The present disclosure is directed to antibodies that bind to DC-HIL on the surface of myeloid-derived suppressor cells, and thus antagonize the T cell suppressor function of these cells, as well as their use in diagnosing and treating cancers such as melanoma.

This application is a continuation of U.S. application Ser. No.15/556,985, filed Sep. 8, 2017, now U.S. Pat. No. 10,517,948, which is anational phase application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2016/021472, filed Mar. 9, 2016, which claimsbenefit of priority to U.S. Provisional Application Ser. No. 62/131,473,filed Mar. 11, 2015, the entire contents of each of which are herebyincorporated by reference.

FEDERAL FUNDING CLAUSE

This invention was made with government support under grant number R01AI064927-05, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention

BACKGROUND 1. Field

The present disclosure relates generally to the fields of medicine,oncology, immunodiagnostics and cancer therapy.

2. Related Art

Skin cancer is the most common primary malignancy in humans, andmelanoma is the type associated with highest mortality. Recent advancesin the treatment of metastatic melanoma involve gene-targeted andimmune-based modalities that have led to some improvement in patientsurvival. A barrier to optimal treatment is the ability of melanoma tocounter anti-tumor T cell response by exploiting host-regulatory systemsincluding regulatory T cells (T_(reg)), tumor-activated (or type 2)macrophages, immature dendritic cells (DC), and myeloid-derivedsuppressor cells (MDSCs) (Frey 2006; Diaz-Montero et al., 2009 andSerafini et al., 2006). Among these immune suppressors, MDSCs stand outbecause of their unsurpassed ability to inhibit T cell function.

MDSCs are distinguished by a CD11b+Gr-1+ phenotype in mice and consistof myeloid progenitor and immature myeloid cells. MDSC can be sortedinto granulocytic and monocytic subtypes (Gabrilovich and Nagaraj, 2009and Movahedi et al., 2008). Because the Gr-1 gene is not found inhumans, CD14+HLA-DR^(no/low) has been used to identify MDSCs in melanomapatients (Filipazzi et al., 2007). MDSCs in healthy individuals are notimmunosuppressive, comprise 30% of BM cells, and differentiate intogranulocytes, macrophages, or DC. By contrast, MDSCs in cancer patientsare strongly immunosuppressive, proliferate exponentially, and theirdifferentiation is blocked by tumor-derived soluble factors (Kusmartsevand Gabrilovich, 2003 and Gabrilovich et al., 1998). Thus, expansion ofMDSC is an endogenous obstacle to successful cancer treatment.

MDSCs have been shown to suppress T cell and natural-killer cellfunctions (Liu et al., 2007), inhibit macrophage cytokine production(Sinha et al., 2007), and induce T_(reg) (Serafini et al., 2008), eitherthrough suppressive soluble mediators like urea/L-ornithine (produced byarginase I) (Rodriguez et al., 2005 and Rodriguez and Ochoa, 2008),nitric oxide (NO) (Bingisser et al., 1998—Nagaraj et al., 2007), and ROS(Kusmartsev and Gabrilovich, 2003), or through coinhibitory pathways(Gabrilovich and Nagaraj, 2009) like CD80/CD86 on MDSC binding to CTLA-4on T cells (Egen et al., 2002 and Yang et al., 2006) and PD-L1 on MDSCsbinding to PD-1 on T cells (Liu et al., 2008 and Liu et al., 2008).Despite these findings, it is controversial which mechanism is criticalto MDSCs suppressive function. This may be due to diversity on phenotypeand even function of MDSCs population in mice and patients withdifferent cancers. Thus, the exact signals responsible for thesuppressive function are not fully understood.

APC's express the DC-HIL receptor (Shikano et al., 2001), also known asglycoprotein nmb (Weterman et al., 1995), osteoactivin (Safadi et al.,2001), and hematopoietic growth factor-inducible neurokinin-1 type (Metzet al., 2007). The inventors have previously demonstrated that DC-HIL onmouse and human APC binds to syndecan-4 (SD-4) on effector/memory (butnot naive) T cells, and such binding inhibits T cell activationtriggered by T cell receptors (TCR) (Chung et al., 2007 and Chung etal., 2009). The DC-HIL/SD-4 pathway curtails T cell-mediatedinflammatory responses in mouse models of contact hypersensitivity andgraft-versus-host disease (Chung et al., 2007 and Akiyoshi et al.,2010). Finally, mouse and human melanoma cells express DC-HIL on theirsurface that utilizes the SD-4 co-inhibitory pathway to suppress T cellsfor fostering melanoma growth (Tomihari et al., 2010). Thus, newreagents to impact the role DC-HIL plays in promoting tumor developmentand immune surveillance would be of great value.

SUMMARY

Thus, in accordance with the present disclosure, there is provided amethod of predicting the progression of melanoma in a subjectcomprising:

(a) obtaining a sample from said subject; and

(b) determining in said sample:

-   -   (i) a DC-HIL level on myeloid-derived suppressor cells (MDSCs)        in said subject; or    -   (ii) the number of DC-HIL-positive MDSCs;        wherein a higher level of DC-HIL on said MDSCs of step (b)(i) as        compared to an MDSC from a non-cancer subject, or a higher        number of DC-HIL-positive MDSCs in step (b)(ii), as compared to        a non-cancer subject, indicates that said subject will have        progression of melanoma. The method may involve performing        steps (a) and (b) a second time and determining a change from        the level or number from the previous assay. The sample may be a        tumor biopsy, or blood or serum. Detection may comprise mass        spectrometry, RT-PCR, or antibody detection, such as ELISA, RIA        or Western blotting. The antibody may be characterized by CDR        sequences as follows:

CDR1- CDR2- CDR3- CDR1- CDR2- CDR3- CLONE L L L H H H 3D5 SSISY TTSHQSSS GYTF INTR TTGF (SEQ (SEQ YPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ(SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 3) 4) 5) 6) 4G5 SSISY TTSHQSSS GYTF INTR TTGF (SEQ (SEQ HPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ(SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 7) 4) 5) 6)The antibody may be a recombinant ScFv (single chain fragment variable)antibody, Fab fragment, F(ab′)₂ fragment, or Fv fragment.

In another embodiment, there is provided a method of predicting theresponse of a subject to melanoma immunotherapy comprising:

-   -   (a) obtaining a sample from said subject; and    -   (b) determining in said sample:        -   (i) a DC-HIL level on myeloid-derived suppressor cell            (MDSCs) in said subject; or        -   (ii) the number of DC-HIL-positive MDSCs;            wherein a lower level of DC-HIL on said MDSCs of step (b)(i)            as compared to a level in a non-cancer subject, or a lower            number of DC-HIL-positive MDSCs in step (b)(ii), as compared            to a non-cancer subject, indicates that said subject will            have a response to melanoma immunotherapy. The method may            involve performing steps (a) and (b) a second time and            determining a change from the level or number from the            previous assay, wherein a lower level of DC-HIL on said            MDSCs of step (b)(i) as compared to a previously measured            level, or a lower number of DC-HIL-positive MDSCs in step            (b)(ii), as compared to a previously measured number,            indicates that said subject is responding to an intervening            melanoma immunotherapy. The sample may be a tumor biopsy, or            blood or serum. Detection may comprise mass spectrometry,            RT-PCR, or antibody detection, such as ELISA, RIA or Western            blotting. The antibody may be characterized by CDR sequences            as follows:

CDR1- CDR2- CDR3- CDR1- CDR2- CDR3- CLONE L L L H H H 3D5 SSISY TTSHQSSS GYTF INTR TTGF (SEQ (SEQ YPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ(SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 3) 4) 5) 6) 4G5 SSISY TTSHQSSS GYTF INTR TTGF (SEQ (SEQ HPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ(SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 7) 4) 5) 6)The antibody may be a recombinant ScFv (single chain fragment variable)antibody, Fab fragment, F(ab′)₂ fragment, or Fv fragment.

In yet another embodiment, there is provided a monoclonal antibody orfragment thereof, wherein the antibody or fragment is characterized byCDR sequences as follows:

CDR1- CDR2- CDR3- CDR1- CDR2- CDR3- CLONE L L L H H H 3D5 SSISY TTSHQSSS GYTF INTR TTGF (SEQ (SEQ YPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ(SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 3) 4) 5) 6) 4G5 SSISY TTSHQSSS GYTF INTR TTGF (SEQ (SEQ HPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ(SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 7) 4) 5) 6)The monoclonal antibody or antibody fragment may be encoded by a lightchain variable sequence according to SEQ ID NOS: 8 or 10 a sequencehaving 70%, 80%, or 90% identity to SEQ ID NOS: 8 or 10, and a heavyvariable chain sequence according to SEQ ID NOS: 9 or 11 or a sequencehaving 70%, 80% or 90% identity to SEQ ID NOS: 9 or 11, respectively.monoclonal antibody or antibody fragment may be encoded by a light chainvariable sequence according to SEQ ID NOS: 8 or 10 a sequence having 95%identity to SEQ ID NOS: 8 or 10, and a heavy variable chain sequenceaccording to SEQ ID NOS: 9 or 11 or a sequence having 95% identity toSEQ ID NOS: 8 or 10, respectively. The monoclonal antibody may compriselight and heavy chain variable region sequences comprising SEQ ID NO: 12and SEQ ID NO: 13, or may comprise light and heavy chain variable regionsequences comprising SEQ ID NO: 14 and 15, respectively. The monoclonalantibody or fragment may be a single chain antibody, a single domainantibody, a chimeric antibody, or a Fab fragment. The monoclonalantibody may be a recombinant antibody having specificity for DC-HIL anda second MDSC surface antigen. The monoclonal antibody or antibodyfragment may be a murine antibody. The monoclonal antibody may be anIgG. The monoclonal antibody may be a humanized antibody. The monoclaonantibody may further comprise an antitumor drug linked thereto. Theantitumor drug may be linked to said antibody through a photolabilelinker, or through an enzymatically-cleaved linker. The antitumor drugmay be a toxin, a radioisotope, a cytokine, or an enzyme. Also providedis a hybridoma expressing any of the monoclonal antibodies or fragmentsset forth above.

In yet another embodiment, there is provided a method of treating cancercomprising administering to said subject an antibody that binds toDC-HIL on the surface of a myeloid-derived suppressor cell (MDSC). Thecancer may be melanoma. The cancer may be lung cancer, brain cancer,head & neck cancer, breast cancer, skin cancer, liver cancer, pancreaticcancer, stomach cancer, colon cancer, rectal cancer, uterine cancer,cervical cancer, ovarian cancer, testicular cancer, or esophagealcancer. The method may further comprise assessing the number ofDC-HIL-positive MDSCs in said subject, and even further comprisesassessing the level of DC-HIL on MDSCs from said subject. The method mayalso further comprise treating said subject with a second anti-canceragent or treatment, such as chemotherapy, radiotherapy, immunotherapy,hormonal therapy, or toxin therapy. The second anti-cancer agent ortreatment may be given at the same time as said antibody, or may begiven before and/or after said antibody. The melanoma may be metastatic,multiply drug resistant or recurrent. The monoclonal antibody orfragment may be a single chain antibody, a single domain antibody, achimeric antibody, or a Fab fragment. The monoclonal antibody may be arecombinant antibody having specificity for DC-HIL and a second MDSCsurface antigen. The monoclonal antibody or antibody fragment may be amurine antibody. The monoclonal antibody may be an IgG. The monoclonalantibody may be a humanized antibody. The antibody may further comprisean antitumor drug linked thereto. The antitumor drug may be linked tosaid antibody through a photolabile linker, or through anenzymatically-cleaved linker. The antitumor drug may be a toxin, aradioisotope, a cytokine, or an enzyme. The antibody or fragment may beconjugated to a bead, a liposome or a nanoparticle, and such bead,liposome or nanoparticle comprises an antitumor drug. The antibody orantibody fragment is characterized by CDR sequences as follows:

CDR1- CDR2- CDR3- CDR1- CDR2- CDR3- CLONE L L L H H H 3D5 SSISY TTSHQSSS GYTF INTR TTGF (SEQ (SEQ YPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ(SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 3) 4) 5) 6) 4G5 SSISY TTSHQSSS GYTF INTR TTGF (SEQ (SEQ HPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ(SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 7) 4) 5) 6)

The monoclonal antibody or antibody fragment may be encoded by a lightchain variable sequence according to SEQ ID NOS: 8 or 10 a sequencehaving 70%, 80%, or 90% identity to SEQ ID NOS: 8 or 10, and a heavyvariable chain sequence according to SEQ ID NOS: 9 or 11 or a sequencehaving 70%, 80% or 90% identity to SEQ ID NOS: 9 or 11, respectively.monoclonal antibody or antibody fragment may be encoded by a light chainvariable sequence according to SEQ ID NOS: 8 or 10 a sequence having 95%identity to SEQ ID NOS: 8 or 10, and a heavy variable chain sequenceaccording to SEQ ID NOS: 9 or 11 or a sequence having 95% identity toSEQ ID NOS: 8 or 10, respectively. The monoclonal antibody may compriselight and heavy chain variable region sequences comprising SEQ ID NO: 12and SEQ ID NO: 13, or may comprise light and heavy chain variable regionsequences comprising SEQ ID NO: 14 and 15, respectively.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The word “about” means plus or minus 5% ofthe stated number.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein. Other objects, features and advantages of the present disclosurewill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIGS. 1A-H. Characterization of DC-HIL-deficient APC. (FIG. 1A) WTallele (consisting of 11 exons) of C57BL/6 background and targeted KOallele are represented schematically. (FIG. 1B) Mouse DNA samples werePCR-amplified with 3 PCR primers (shown on the map): (1) intron betweenexons 1 and 2 (669 bp PCR band by primers #1 and #2); and (2) regionspanning the intron to Neo gene (952 bp by primers #1 and #3), andseparated on 1.5% agarose gel. WT and KO allele produces bands of 669and 952 bp, respectively; heterozygote showed a mixed pattern. (FIG. 1C)Total RNA isolated from BM-derived DC of a KO or WT mouse was analyzedby RT-PCR using 2 primer sets to amplify exons 2-3 (E2-3, 416 bp) andexons 5-9 (E5-9, 673 bp). β-actin mRNA was also PCR-amplified. (FIG. 1D)BM-DC were used to immunoblot DC-HIL and b-actin proteins (20 mg ofcrude protein/lane) using 1E4 anti-DC-HIL and anti-b-actin Ab. Totalproteins were also stained by Coomassie blue. (FIGS. 1E-F) BM-DC ormacrophages (MF) from WT or KO mice were examined by FACS for expressionof DC-HIL and CD11c⁺ (FIG. 1E) or CD11b (FIG. 1F). (FIG. 1G) Varyingnumbers of BM-DC from WT or KO mice were cocultured with CD4⁺ or CD8⁺ Tcells (from OT-II or OT-I transgenic mice, respectively) with OVApeptide, and IL-2 and/or IFN-g secretion measured. (FIG. 1H) DCpreparations were assayed for surface expression of CD80 and CD86 onCD11c⁺ cells. Two other KO and WT mice showed similar results.*Students' t-test (p<0.001) between WT and KO.

FIGS. 2A-D. Growth and metastasis of B16 melanoma are suppressed inDC-HIL^(−/−) mice. (FIG. 2A) Tumor volume of B16 cells implanted into WTor DC-HIL KO mice (n=5). (FIG. 2B) Tumor volume after implantingDC-HIL-KD-B16 cells (n=5). Lung metastasis (FIG. 2C) at 19 days afterB16 cells injected i. v. into WT or KO mice (n=10); lung weight, numberof metastatic foci, melanin content/lung, and melanin content/focusplotted (FIG. 2D). Representative data from 3 separate experiments,*p<0.01 versus WT.

FIGS. 3A-E. Melanoma induces DC-HIL expression by most potent MDSCsuppressors. (FIG. 3A) Splenocytes from B16 melanoma-bearing ortumor-free mice (n=3) were assayed for % DC-HIL⁺ cells in 3 myeloidpopulations. (FIGS. 3B-C) MDSC isolated from mice with (FIG. 3B) orwithout (FIG. 3C) melanoma were examined for expression of Gr1 andcoinhibitory receptors (%). (FIG. 3D) These myeloid cells (increasingcell ratios) were cocultured with CFSE-labeled T-cells activated byanti-CD3/CD28 Ab. T-cell proliferation (%) was determined by FACS(histograms). (FIG. 3E) Purified myeloid cells were examined for Tcell-stimulatory capacity. Different numbers of myeloid cells werepulsed with gp100 Ag and added to culture of CD8⁺ pmel-1 T-cells.Culture of T-cells with Ag served as control (No). T-cell proliferationwas measured by ³H-thymidine (TdR) incorporation. *p<0.01.

FIGS. 4A-E. DC-HIL is expressed by some MDSC from several tissues. Threeweeks after implanting B16 melanoma cells in mice, CD11b⁺Gr1⁺ MDSC werepurified from different tissues and examined for DC-HIL expression byFACS. (FIG. 4A) BM cells were prepared from the femur, from which MDSCwere purified and fluorescently stained with anti-Gr1 and anti-DC-HILmAb (or control IgG). (FIG. 4B) Peripheral blood was collected frommouse tail veins. (FIG. 4C) Tumor-infiltrating cells were prepared fromB16 tumor (˜2 cm³): The tumor was minced in PBS, treated with digestiveenzymes, and applied to Ficoll-gradient to remove debris. % DC-HIL⁺cells among total MDSC cells are shown in dot-plots. (FIG. 4D) MDSCpurified from splenocytes of mice with B16 melanoma were analyzed forexpression of Gr-1 vs. Ly6C. Based on differential expression of thesemarkers, MDSC sorted into 4 different fractions: Fr. 1(Ly6C^(high)Gr1^(low)); Fr. 2 (Ly6C^(med)Gr1^(high)); Fr. 3(Ly6C^(med)Gr1^(low)); and Fr. 4 (Ly6C^(low)Gr1^(low)). Each fractionwas examined for DC-HIL expression: open and gray-filled histograms showanti-DC-HIL mAb and control IgG staining, respectively. Fr. 1 showedhighest expression of DC-HIL. (FIG. 4E) MDSC subsets (Fr. 1 through Fr.4) were purified by FACS sorting and cocultured with pmel-1splenocytes/Ag at different cell ratios. T cell activation was assessedby proliferation, and the suppressive ability of each Fr orunfractionated MDSC is expressed as % suppression (1−cpm of MDSC-addedculture/cpm of T cell alone ×100%). *p<0.001 between Fr.1 and Fr.2. Dataare representative of 3 experiments.

FIGS. 5A-H. DC-HIL mediates T cell-suppressor activity of MDSC. (FIGS.5A-B) MDSC from melanoma-bearing mice were cocultured with pmel-1splenocytes (Spl), gp100 Ag, and anti-DC-HIL mAb (FIG. 5A),anti-coinhibitor Ab or control IgG (FIG. 5B). ³H-TdR incorporationmeasured. (FIG. 5C) Undepleted (DC-HIL⁺) or DC-HIL-depleted(DC-HIL^(neg)) MDSC were assayed for suppression of pmel-1 splenocyteproliferation triggered by Ag (increasing ratios). (FIG. 5D) Mice (n=5)injected with pmel-1 CD8⁺ T-cells and DC-HIL⁺ MDSC or DC-HIL^(neg) MDSC.Ten days after giving gp100, IFN-g-producing cells in LN were counted.(FIG. 5E) Tumor growth following coinjection of DC-HIL⁺ or DC-HIL^(neg)MDSC with B16 cells s.c. into naive mice (n=5). Using similar methods,DC-HIL^(−/−)-MDSC were compared with DC-HIL^(+/+) counterparts forDC-HIL expression by FACS (FIG. 5F), T-cell suppressing (FIG. 5G) andtumor-promoting ability (CD11b^(neg) cells as control) (FIG. 5H).*p<0.01.

FIGS. 6A-B. DC-HIL^(−/−) MDSC are unable to induce expression of IFN-γand iNOS following DC-HIL-crosslinking. MDSC isolated from WT or DC-HILKO mice (n=3) bearing melanoma were DC-HIL-crosslinked and assayed (FIG.6A) for IFN-γ production (by ELISA) and (FIG. 6B) for iNOS mRNAexpression (by real time PCR): the former was measured individually byMDSC (mean±sd, n=3); and the latter by pooled MDSC. Data are shown asfold increase (anti-DC-HIL-treated culture vs. control IgG). Data arerepresentative of 2 experiments. *p<0.001 between WT and KO.

FIGS. 7A-I. Crosslinked DC-HIL on MDSC induces tyrosine phosphorylationand IFN-γ/iNOS expression. (FIGS. 7A-C) MDSC cocultured with pmel-1splenocytes (1:1 ratio) with inhibitors; including (FIG. 7A)anti-cytokine Ab; (FIG. 7B) 5 mM L-N^(G)-monomethyl-arginine citrate(NOSs); 0.5 mM N⁶-(1-iminoethyl)-L-lysine (NOS-2); 1 mMN-hydroxyl-nor-arginine (Arg); 0.2 mM 1-methyl-tryptophan (Indol); 1000U/ml catalase (C-ROS); and 200 U/ml superoxide dismutase (S-ROS); and(FIG. 7C) anti-DC-HIL mAb or DC-HIL-Fc. ³H-TdR uptake was measured.(FIG. 7D) MDSC cocultured with SD-4^(+/+) or SD-4^(−/−) pmel-1splenocytes. (FIGS. 7E-I) At varying times after crosslinking with Ab,MDSC were assayed for: tyrosine-phosphorylation (p-Tyr) on DC-HILprotein (FIG. 7E); cytokine mRNA and secretion (FIGS. 7F-G); mRNA of NOSgenes (FIG. 7H); or NO production (FIG. 7I). Data (mean±sd, n=3) areshown as fold increase relative to control. *p<0.01.

FIGS. 8A-F. Infusion of anti-DC-HIL mAb suppresses melanoma growth andexpansion of MDSC. (FIG. 8A) Tumor volume 6 days after implanting B16cells into WT mice (n=7), mice injected with anti-DC-HIL mAb or controlIgG on indicated days (closed arrows). On days shown by gray arrows inFIG. 8A, blood taken from mouse, MDSC counted from PBMCs by FACS (FIG.8B), and data summarized (FIG. 8C). (FIG. 8D) A day after 3 injections,IFN-g-secreting cells in spleen or LN in each mouse (n=3) counted asnumber per 1×10⁴ cells. (FIG. 8E) Tumor volume on mice treated with the2 Abs 11 days after implanting KD-B16 cells (n=7). (FIG. 8F) Tumorvolume on DC-HIL^(−/−) mice treated with Ab 7 days after co-injection ofB16 and MDSC. *p<0.001.

FIGS. 9A-B. Amino acid sequences of 3D5 mouse anti-human DC-HIL mAb.Total RNA isolated from 3D5-producing B cells is reverse-transcribed tocDNAs and PCR-amplified with primers: (1) for V_(H) region,5′-RCTACAGGTGTCCACTCC-3′ (encoding the signal peptide; SEQ ID NO: 20)and 5′-TAGCCCTTGACCAGGCATCC-3′ (the CH region; SEQ ID NO: 21); and (2)for Vk region, 5′-TCAGCTTCYTGCTAATCAGTG-3′ (the signal peptide; SEQ IDNO: 22) and 5′-TGGTGGGAAGATGGATACAG-3′ (the Ck region; SEQ ID NO: 23).Resulting PCR product was ligated to pCR2.1 vector, and the insert wasdetermined for DNA sequences, from of which amino acid sequences werededuced. CDR sequences (shown in green) in the V_(H) (FIG. 9A) and Vk(FIG. 9B) regions were determined using IMGT programs. (A,heavy-chain=SEQ ID NO: 9; B, light-chain=SEQ ID NO: 8)

FIG. 10A-B. Amino acid sequences of 4G5 mouse anti-human DC-HIL mAb.Total RNA isolated from 4G5-producing B cells is reverse-transcribed tocDNAs and PCR-amplified with primers: (1) for V_(H) region,5′-RCTACAGGTGTCCACTCC-3′ (encoding the signal peptide; SEQ ID NO: 20)and 5′-TAGCCCTTGACCAGGCATCC-3′ (the CH region; SEQ ID NO: 21); and (2)for Vk region, 5′-TCAGCTTCYTGCTAATCAGTG-3′ (the signal peptide; SEQ IDNO: 22) and 5′-TGGTGGGAAGATGGATACAG-3′ (the Ck region; SEQ ID NO: 23).Resulting PCR product was determined for DNA and amino acid sequences.CDR sequences in the V_(H) (FIG. 10A) and Vk (FIG. 10B) regions areshown in green. All amino acid sequences are identical to 3D5, but witha mutation of Y→H (shown in red) in the CDR3 of the Vk region. (A,heavy-chain=SEQ ID NO: 11; B, light-chain=SEQ ID NO: 10)

FIGS. 11A-C. Characterization of 3D5 and 4G5 mAb. (FIG. 11A) Specificityof 3D5 mAb to DC-HIL: protein or total cell extracts were subjected toSDS-PAGE/immunoblotting analysis with 1 mg/ml. DC-HIL-Fc (a recombinantprotein consisting of the extracellular domain of DC-HIL fused to the Fcportion of Ig), mIgG (mouse IgG, a control for DC-HIL-Fc) and cellextracts from COS-1 cells transfected with a empty vector or DC-HILgene, or from varying human cell lines (embryonal fibroblast 293T,melanoma SK-MEL-28, and primary cultured melanocytes). Closed and openarrow heads indicate DC-HIL proteins and nonspecific bands,respectively. (FIG. 11B) Immunoblotting with 4G5 mAb. (FIG. 11C)SK-MEL-28 cells were stained with control IgG (shown in closedhistograms), 3D5 or 4G5 mAb (10 mg/ml, open histograms) and analyzed forexpression of surface-DC-HIL by FACS. MIF means mean fluorescenceintensity.

FIGS. 12A-D. Positive correlation between bloodDC-HIL⁺CD14⁺HLA-DR^(no/low) myeloid-derived suppressor cells (MDSCs) andmelanoma stage. Peripheral blood mononuclear cells (PBMCs) from melanomapatients (stages 0-IV, referred to FIG. 5) or dysplastic nevus (DN), andfrom healthy donors (HD) were analyzed for CD14 vs. HLA-DR expression,in which CD14⁺HLA-DR^(no/low) MDSCs are indicated (%). These cells wereFACS-gated and examined for expression of DC-HIL vs. CD14. Data shownare representative of each group (FIG. 12A). % CD14⁺HLA-DR^(no/low)MDSCs (FIG. 12B) or % DC-HIL⁺ MDSCs/PBMC (FIG. 12C) in each cohort issummarized (mean %±sd). Statistical significance for each stage wascalculated by comparison with HD. (FIG. 12D) % blood DC-HIL⁺ MDSCs/PBMCswas assayed at indicated times post-resection in 9 patients with stage 0melanoma (data for patient M71 are in red). Detailed data are referredto FIGS. 14 and 15. *p<0.001 and **p<0.01. Note that DC-HIL expressionwas assayed using 3D5 anti-DC-HIL mAb.

FIG. 13. Melanoma patients and healthy controls used for study. Theinventors recruited melanoma patients staged clinically based onAmerican Joint Committee on Cancer criteria: metastasis to distant organ(stage IV); lymph node metastasis (stage III); limited to skin withvarying thickness (stage 0-II). Patients with dysplastic nevi (atypicalbut not malignant histological changes) and healthy donors served asnegative controls.

FIG. 14. Frequency of blood CD14⁺HLA-DR^(no/low) MDSCs in melanomapatients after resection. All patients were diagnosed for melanoma insitu (MIS, stage 0). At indicated months after resection, PBMCs isolatedfrom patients were analyzed for immunological features. “0 month” meanstime of resection. M71 patient was found to have a second MIS 3 monthsafter resection of the first melanoma.

FIG. 15. Blood CD14⁺HLA^(no/low) MDSCs at sequential blood draws fromhealthy controls. Blood from healthy controls was drawn at indicatedmonths and analyzed for immunological features.

FIG. 16. Expression of DC-HIL on four different MDSC subsets in melanomapatients. PBMCs isolated from a patient with melanoma of stage III wereFACS analyzed for frequency of 4 different MDSC subsets (shown inred-lined box) and for DC-HIL expression: CD14⁺HLA-DR^(no/low)CD11b⁺cells represent at 2.2% in total PBMCs with 97% DC-HIL⁺ cells in thesubset; CD14⁺IL-4Ra⁺ at 0.5% with 74% DC-HIL; CD14^(neg)CD11b⁺CD15⁺ at0.2% with 31% DC-HIL; and CD14^(neg)IL-4Ra⁺CD15⁺ at 0.003% with 37%DC-HIL. Second melanoma patient showed similar results. Note that DC-HILexpression was measured using 3D5 anti-DC-HIL mAb.

FIGS. 17A-D. 3D5 anti-DC-HIL mAb treatment restored IFN-γ response ofT-cells from melanoma patients. (FIG. 17A) CD14⁺HLA-DR^(no/low) MDSCsfrom a stage III patient or a healthy donor co-cultured with autologousT-cells/HLA-DR⁺ cells (mixed with 1:1 ratio) at varying cell ratios withanti-CD2/CD3/CD28 Ab. After culturing for 5 days, IFN-γ secretion wasmeasured (mean±sd, n=3). Representative data of 3 different patients.(FIG. 17B) Increasing doses of 3D5 mAb or control IgG were added to thecoculture (1:1 cell ratio). (FIG. 17C) PBMCs from same patients withstages III/IV (n=25) were cultured for 5 days with 3D5 mAb or controlIgG (20 mg/ml). IFN-γ amounts were assayed by ELISA, and fold increaseby mAb vs. IgG is shown with Pearson's correlation coefficient r. (FIG.17D) Same experiments were performed with all samples, and values of-fold increase in IFN-γ production plotted to cancer stage. *p<0.001.

FIG. 18. Effect of 4G5 anti-DC-HIL mAb on T cell-suppressive activity ofMDSCs. CD14⁺HLA-DR^(neg) MDSCs were purified from a melanoma patientwith Stage III and subjected to the T cell suppression assay shown inFIG. 17B, with a cell ratio of 1:1 (T cells: MDSCs) and 20 mg/ml of mIgGor 4G5 mAb. IFN-γ secretion was measured by ELISA. “ns' stands for notstatistically significant.

FIG. 19. Expansion of DC-HIL⁺ MDSCs in blood of patients withnon-melanoma cancers. CD14⁺HLA-DR^(no/low) MDSCs were determined for %in blood of patients with advanced cancers, including squamous cellcarcinoma (SCC), breast cancer, colon or lung cancers, and plotted(mean±SD). Most lung cancers are non-small cell adenocarcinoma (9 out of10). The range of healthy controls' values (HD, n=25) is shown in thegraph. Note that DC-HIL expression was assayed using 3D5 anti-DC-HILmAb.

FIG. 20. Nucleic acid sequences for variable regions. SEQ ID NOS: 12-15are shown.

FIGS. 21A-B. T cell suppressor activity of MDSC isolated from pancreaticcancer patient. MDSC were purified from blood of a patient withmetastatic pancreatic cancer: PBMCs were isolated from the blood,depleted of HLA-DR⁺ cells, and sorted into CD14⁺ and CD14^(neg)fractions using Ab-coated magnetic beads: The former contains MDSC at85-90% and the latter T cells at ˜85%. (FIG. 21A) These cells were mixedat varying cell ratios with beads coated with anti-CD2/CD3/CD28 Ab (foractivating T cells). 3D5 anti-DC-HIL mAb or control IgG (25 mg/ml) wereadded to some of 1:1 cocultures. After culturing for 5 days, IFN-gamounts in the culture supernatant were determined by ELISA (Data areshown by mean±SD, n=3). (FIG. 21B) On day 3 after culturing, culture ofT cell alone and cocultures (1:1 cell ratio) with control IgG or 3D5 (25mg/ml) were observed under microscope (10× magnification). Aggregatesshow T cell activation. Data shown are representative of T cell assayswith MDSC from 4 different patients. Note that 3D5 mAb restored T cellresponse of pancreatic patients almost completely.

FIG. 22. T cell suppressor activity of MDSC from colon cancer patients.MDSC were purified from blood of patients (#14 and #15) with metastaticcolon cancer (FIGS. 21A-B). T cells and MDSC from the same patient weremixed at a cell ratio of 1:1 with beads coated with anti-CD2/CD3/CD28Ab. As control, T cell alone culture was set. Varying doses of 3D5anti-DC-HIL mAb or control IgG were added to 1:1 cocultures: 0 meansjust 1:1 cocultures. Five days later, IFN-g amounts were assayed byELISA (mean±SD, n=3). Data shown are representative of T cell assayswith MDSC from 6 different patients. NS means “not significant” between50 mg/ml 3D5 and T cell alone. Note that 3D5 mAb restored T cellresponse of colon patients almost completely.

FIGS. 23A-B. T cell suppressor activity of MDSC isolated from kidneycancer patient. MDSC were purified from blood of a patient withmetastatic kidney cancer (FIGS. 21A-B). T cells and MDSC from the samepatient were mixed at varying cell ratios with beads coated withanti-CD2/CD3/CD28 Ab. 3D5 anti-DC-HIL mAb or control IgG (25 mg/ml) wereadded to some of 1:1 cocultures. On day 5, IFN-g amounts in the culturesupernatant were assayed by ELISA. Data shown are representative of Tcell assays with MDSC from 2 different patients. Note that 3D5 mAbrestored T cell response of kidney patients by 20-fold, compared toIgG-treated culture.

FIGS. 24A-B. T cell suppressor activity of MDSC isolated from BCC andSCC cancer patients. MDSC were purified from blood of a patient withskin cancer (basal cell carcinoma, BCC; or squamous cell carcinoma, SCC)as described in FIGS. 21A-B. T cells and MDSC from the same patient weremixed at varying cell ratios with beads coated with anti-CD2/CD3/CD28Ab. 3D5 anti-DC-HIL mAb or control IgG (25 mg/ml) were added to some of1:1 cocultures. After culturing for 5 days, IFN-g secretion was assayedby ELISA. Data shown are representative of T cell assays with MDSC from2 different patients in each skin cancer. Note that 3D5 mAb restored Tcell response of BCC patients completely, but no significant effect onSCC.

FIG. 25. DC-HIL⁺ MDSC blood levels in varying cancer types. Bloodsamples freshly isolated from varying cancer patients (n; total casestested) were determined for % MDSC/PBMC, % DC-HIL+ MDSC/total MDSC, and% DC-HIL+ MDSC/PBMC. *Median and the range are shown. **Gastro-esophageal junction.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Here, the inventors report that DC-HIL is expressed markedly inmacrophages, DC and MDSCs in mice bearing melanoma, but has little to noexpression in tumor-free mice. Among those 3 myeloid cells, MDSCs werethe most expanded and most potent in suppressing T cell activation,prompting them to probe a role for DC-HIL+ MDSCs in melanoma growth. Theinventors found DC-HIL to be the only co-inhibitory ligand, and IFN-γand NO the only soluble mediators, required for MDSCs suppressoractivity. Ligation of DC-HIL on MDSCs induced tyrosine phosphorylationof DC-HIL's intracellular domain, which then triggered the IFN-γ/NOS-2pathway (Kamijo et al., 1994). Deficient DC-HIL gene expression markedlyreduced melanoma growth and metastasis in mice, and blocking DC-HILfunction by anti-DC-HIL mAb infusion into melanoma-bearing miceinhibited tumor growth and prevented MDSC expansion. These beneficialoutcomes resulted from negation of the suppressive function of DC-HIL+MDSCs (but not of DC-HIL+ melanoma nor DC-HIL+ APC).

The inventors also found DC-HIL to be induced in circulating MDSCs ofmetastatic melanoma patients, and that DC-HIL is required for MDSCs tosuppress T cells. These results indicate that DC-HIL is the criticalmediator of MDSCs deleterious effects in melanoma and that DC-HIL+ MDSCscan be targeted to improve melanoma immunotherapy. In furtherance ofthis hypothesis, the inventors have developed monoclonal antibodiesagainst DC-HIL and found that they can restore activity of T-cells fromcancer patients. These and other aspects of the disclosure are describedin detail below.

I. MDSCS AND DC-HIL

A. MDSCs

MDSCs (myeloid-derived suppressor cells) are a heterogenous populationof immune cells from the myeloid lineage (a cluster of different celltypes that originate from bone marrow stem cells), to which dendriticcells, macrophages and neutrophils also belong. However, MDSCs possessstrong immunosuppressive activities rather than immunostimulatoryproperties. Myeloid cells interact with T cells (the effector immunecells that kill pathogens, infected and cancer cells) to regulate theirfunctions, some of which are still under heated debate and closeexamination by the scientific community.

MDSCs are usually defined in mouse models as myeloid cells expressinghigh levels of CD11b and GR1, which exhibit potent T cell inhibitoryactivities. In human, MDSCs are generally defined as expressing highlevels of some characteristic markers such as CD33, CD11b and low levelsof HLA DR. However, it remains to be resolved as there is nointernational consensus on how human subsets of MDSCs should be defined.

Generally speaking, regardless of whether they are from mice or human,MDSCs suppressor function lies in their ability to inhibit T cellproliferation and activation. In healthy individuals, immature myeloidcells formed in the bone marrow differentiated to dendritic cells,macrophages and neutrophils. However, under chronic inflammatoryconditions (viral and bacterial infections) or cancer, myeloiddifferentiation is skewed towards the expansion of MDSCs. These MDSCsinfiltrate inflammation sites and tumors, where they stop immuneresponses by inhibiting T cells and NK cells, for example. MDSCs alsoaccelerate angiogenesis, tumor progression and metastasis. Therefore,they have become a key therapeutic target. Clinical and experimentalevidence has shown that cancer tissues with high infiltration of MDSCare associated with poor patient prognosis and resistance to therapies.

Cytokines are key signals involved in the generation of MDSCs. Tumorcell lines overexpressing colony stimulating factors (e.g., G-CSF andGM-CSF) have long been used in in vivo models of MDSC generation.GM-CSF, G-CSF and IL-6 allow the in vitro generation of MDSCs thatretain their suppressive function in vivo. In addition to CSF, othercytokines such as IL-6, IL-10, VEGF, PGE2 and IL-1 have been implicatedin the development and regulation of MDSCs. The myeloid-differentiationcytokine GM-CSF is a key factor in MDSC production from bone marrow, andit has been shown that the c/EBP(3 transcription factor plays a key rolein the generation of in vitro bone marrow-derived and in vivotumor-induced MDSCs. Moreover, STAT3 promotes MDSC differentiation andexpansion and IRF8 has been suggested to counterbalance MDSC-inducingsignals.

MDSC activity was originally described as suppressors of T cells, inparticular of CD8+ T-cell responses. The spectrum of action of MDSCactivity also encompasses NK cells, dendritic cells and macrophages.Suppressor activity of MDSC is determined by their ability to inhibitthe effector function of lymphocytes. Inhibition can be caused differentmechanisms. It is primarily attributed to the effects of the metabolismof L-arginine. Another important factor influencing the activity ofMDSCs is oppressive ROS.

In addition to host-derived factors, pharmacologic agents also haveprofound impact on MDSCs. Chemotherapeutic agents belonging to differentclasses have been reported to inhibit MDSCs. Although this effect maywell be secondary to inhibition of hematopoietic progenitors, there maybe grounds for search of selectivity based on long-known differentialeffects of these agents on immunocompetent cells and macrophages.

B. DC-HIL

Murine DC-HIL has a leader sequence (aa 1-19), a long extracellulardomain (ECD, aa 20-499), a transmembrane domain (aa 500-523), and acytoplasmic domain (aa 524-574). The ECD contains 11 potentialN-glycosylation sites (NX(S/T)) and several putative O-glycosylationsites based on the stretch of proline-, serine-, and threonine-richregion, and a proline-rich region (aa 320-352) that presumably forms ahinge, as seen in proteins like IgA, which can mediate protein-proteininteractions. Other functional motifs are an RGD sequence (aa 64-66), anintegrin-binding sequence, and a KRFR (SEQ ID NO: 16) sequence (aa23-26) that matches a heparin-binding motif composed of a stretch ofbasic residues (BBXB, where B represents a basic residue). Thecytoplasmic tail contains an immunoreceptor tyrosine-based activationmotif (ITAM (SEQ ID NO: 17), YXXI (SEQ ID NO: 18), aa 529-532, where Xrepresents all other amino acid residues) and two lysosomal targetingdi-leucine motifs (LL, aa 548-549 and 566-567).

Human DC-HIL has a leader sequence (aa 1-19), a long extracellulardomain (ECD, aa 20-495), a transmembrane domain (aa 496-518), and acytoplasmic domain (aa 519-572). The ECD contains 11 potentialN-glycosylation sites (NX(S/T)) and several putative O-glycosylationsites based on the stretch of proline-, serine-, and threonine-richregion, and a proline-rich region (aa 320-349) that presumably forms ahinge, as seen in proteins like IgA, which can mediate protein-proteininteractions. Other functional motifs are an RGD sequence (aa 64-66), anintegrin-binding sequence, and a KRFH (SEQ ID NO: 19) sequence (aa23-26) that matches a heparin-binding motif composed of a stretch ofbasic residues (BBXB, where B represents a basic residue). Thecytoplasmic tail contains an immunoreceptor tyrosine-based activationmotif (ITAM (SEQ ID NO: 17), YXYI (SEQ ID NO: 32), aa 525-528) and twolysosomal targeting di-leucine motifs (LL, aa 516-517 and 562-563).

Previously, the inventors identified DC-HIL as a highly glycosylatedtype I transmembrane protein of 125 and 95 kDa containing anextracellular Ig-like domain (Shikano et al., 2001). They also showedthat DC-HIL is expressed constitutively at high levels on the surface ofall dendritic cell subsets, including plasmacytoid dendritic cells andLangerhans cells and at lower levels on macrophages (Shikano et al.,2001), and that its expression can be induced in non-lymphoid cells(keratinocytes) following IFN-γ treatment. In human, DC-HIL is expressedconstitutively at high levels by CD14⁺ monocytes and dendritic cells(but not by other leukocytes). They have also shown that DC-HIL is anegative regulator of T-cell activation (Chung et al., 2007a; Chung etal., 2007b) through binding to syndecan-4 on activated T-cells,indicating that interaction of DC-HIL with syndecan-4 attenuates T-cellactivation triggered by anti-CD3 Ab or by APCs in a manner resemblingthe inhibitory function of PD-L1/PD-L2.

II. PRODUCING MONOCLONAL ANTIBODIES

A. General Methods

It will be understood that monoclonal antibodies binding to DC-HIL willhave several applications. These include the production of diagnostickits for use in detecting and diagnosing cancer, as well as for cancerimmunosuppression and cancer therapies. In these contexts, one may linksuch antibodies to diagnostic or therapeutic agents, use them as captureagents or competitors in competitive assays, or use them individuallywithout additional agents being attached thereto. The antibodies may bemutated or modified, as discussed further below. Methods for preparingand characterizing antibodies are well known in the art (see, e.g.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;U.S. Pat. No. 4,196,265).

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies. Thefirst step for both these methods is immunization of an appropriate hostor identification of subjects who are immune due to prior naturalinfection. As is well known in the art, a given composition forimmunization may vary in its immunogenicity. It is often necessarytherefore to boost the host immune system, as may be achieved bycoupling a peptide or polypeptide immunogen to a carrier. Exemplary andpreferred carriers are keyhole limpet hemocyanin (KLH) and bovine serumalbumin (BSA). Other albumins such as ovalbumin, mouse serum albumin orrabbit serum albumin can also be used as carriers. Means for conjugatinga polypeptide to a carrier protein are well known in the art and includeglutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester,carbodiimyde and bis-biazotized benzidine. As also is well known in theart, the immunogenicity of a particular immunogen composition can beenhanced by the use of non-specific stimulators of the immune response,known as adjuvants. Exemplary and preferred adjuvants include completeFreund's adjuvant (a non-specific stimulator of the immune responsecontaining killed Mycobacterium tuberculosis), incomplete Freund'sadjuvants and aluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster injection, also may be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate MAbs.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens or lymph nodes, or from circulating blood. Theantibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized or human or human/mousechimeric cells. Myeloma cell lines suited for use in hybridoma-producingfusion procedures preferably are non-antibody-producing, have highfusion efficiency, and enzyme deficiencies that render then incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions. Oneparticular murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline. More recently, additional fusion partner lines for use with humanB cells have been described, including KR12 (ATCC CRL-8658; K6H6/B5(ATCC CRL-1823 SHM-D33 (ATCC CRL-1668) and HMMA2.5 (Posner et al.,1987). The antibodies in this disclosure were generated using theSP2/0/mIL-6 cell line, an IL-6 secreting derivative of the SP2/0 line.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods also is appropriate (Goding, pp.71-74, 1986). Fusion procedures usually produce viable hybrids at lowfrequencies, about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, infused cells (particularly the infused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine. Ouabain is added if the B cell source isan Epstein Barr virus (EBV) transformed human B cell line, in order toeliminate EBV transformed lines that have not fused to the myeloma.

The preferred selection medium is HAT or HAT with ouabain. Only cellscapable of operating nucleotide salvage pathways are able to survive inHAT medium. The myeloma cells are defective in key enzymes of thesalvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT),and they cannot survive. The B cells can operate this pathway, but theyhavea limited life span in culture and generally die within about twoweeks. Therefore, the only cells that can survive in the selective mediaare those hybrids formed from myeloma and B cells. When the source of Bcells used for fusion is a line of EBV-transformed B cells, as here,ouabain is also used for drug selection of hybrids as EBV-transformed Bcells are susceptible to drug killing, whereas the myeloma partner usedis chosen to be ouabain resistant.

Culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays dot immunobindingassays, and the like. The selected hybridomas are then serially dilutedor single-cell sorted by flow cytometric sorting and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into an animal (e.g., amouse). Optionally, the animals are primed with a hydrocarbon,especially oils such as pristane (tetramethylpentadecane) prior toinjection. When human hybridomas are used in this way, it is optimal toinject immunocompromised mice, such as SCID mice, to prevent tumorrejection. The injected animal develops tumors secreting the specificmonoclonal antibody produced by the fused cell hybrid. The body fluidsof the animal, such as serum or ascites fluid, can then be tapped toprovide MAbs in high concentration. The individual cell lines could alsobe cultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations. Alternatively, human hybridoma cells lines can be usedin vitro to produce immunoglobulins in cell supernatant. The cell linescan be adapted for growth in serum-free medium to optimize the abilityto recover human monoclonal immunoglobulins of high purity.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asFPLC or affinity chromatography. Fragments of the monoclonal antibodiesof the disclosure can be obtained from the purified monoclonalantibodies by methods which include digestion with enzymes, such aspepsin or papain, and/or by cleavage of disulfide bonds by chemicalreduction. Alternatively, monoclonal antibody fragments encompassed bythe present disclosure can be synthesized using an automated peptidesynthesizer.

It also is contemplated that a molecular cloning approach may be used togenerate monoclonals. For this, RNA can be isolated from the hybridomaline and the antibody genes obtained by RT-PCR and cloned into animmunoglobulin expression vector. Alternatively, combinatorialimmunoglobulin phagemid libraries are prepared from RNA isolated fromthe cell lines and phagemids expressing appropriate antibodies areselected by panning using viral antigens. The advantages of thisapproach over conventional hybridoma techniques are that approximately10⁴ times as many antibodies can be produced and screened in a singleround, and that new specificities are generated by H and L chaincombination which further increases the chance of finding appropriateantibodies.

Other U.S. patents, each incorporated herein by reference, that teachthe production of antibodies useful in the present disclosure includeU.S. Pat. No. 5,565,332, which describes the production of chimericantibodies using a combinatorial approach; U.S. Pat. No. 4,816,567 whichdescribes recombinant immunoglobulin preparations; and U.S. Pat. No.4,867,973 which describes antibody-therapeutic agent conjugates.

B. Antibodies of the Present Disclosure

Antibodies according to the present disclosure may be defined, in thefirst instance, by their binding specificity, which in this case is forDC-HIL on the surface of MDSCs. Those of skill in the art, by assessingthe binding specificity/affinity of a given antibody using techniqueswell known to those of skill in the art, can determine whether suchantibodies fall within the scope of the instant claims. In one aspect,there is provided a monoclonal antibody having CDRs as defined below:

CDR1- CDR2- CDR3- CDR1- CDR2- CDR3- CLONE L L L H H H 3D5 SSISY TTSHQSSS GYTF INTR TTGF (SEQ (SEQ YPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ(SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 3) 4) 5) 6) 4G5 SSISY TTSHQSSS GYTF INTR TTGF (SEQ (SEQ HPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ(SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 7) 4) 5) 6)Such antibodies may be produced by the clones discussed below in theExamples section using methods described herein.

In a second aspect, the antibodies may be defined by their variablesequence, which include additional “framework” regions. These areprovided in SEQ ID NOS: 8-11 (protein) and SEQ ID NOS: 12-15 (nucleicacids). Furthermore, the antibodies sequences may vary from thesesequences, optionally using methods discussed in greater detail below.For example, nucleic acid sequences may vary from those set out above inthat (a) the variable regions may be segregated away from the constantdomains of the light and heavy chains, (b) the nucleic acids may varyfrom those set out above while not affecting the residues encodedthereby, (c) the nucleic acids may vary from those set out above by agiven percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids may vary fromthose set out above by virtue of the ability to hybridize under highstringency conditions, as exemplified by low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.15 MNaCl at temperatures of about 50° C. to about 70° C., (e) the aminoacids may vary from those set out above by a given percentage, e.g.,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology,or (f) the amino acids may vary from those set out above by permittingconservative substitutions (discussed below). Each of the foregoingapply to the nucleic acid sequences set forth as SEQ ID NOS: 12-15, andthe amino acid sequences of SEQ ID NOS: 8-11.

C. Engineering of Antibody Sequences

In various embodiments, one may choose to engineer sequences of theidentified antibodies for a variety of reasons, such as improvedexpression, improved cross-reactivity, diminished off-target binding orabrogation of one or more natural effector functions, such as activationof complement or recruitment of immune cells (e.g., T cells). Thefollowing is a general discussion of relevant techniques for antibodyengineering.

Hybridomas may be cultured, then cells lysed, and total RNA extracted.Random hexamers may be used with RT to generate cDNA copies of RNA, andthen PCR performed using a multiplex mixture of PCR primers expected toamplify all human variable gene sequences. PCR products can be clonedinto pGEM-T Easy vector, then sequenced by automated DNA sequencingusing standard vector primers. Assay of binding and neutralization maybe performed using antibodies collected from hybridoma supernatants andpurified by FPLC, using Protein G columns.

Recombinant full length IgG antibodies were generated by subcloningheavy and light chain Fv DNAs from the cloning vector into a LonzapConIgG1 or pConK2 plasmid vector, transfected into 293 Freestyle cellsor Lonza CHO cells, and antibodies were collected and purified from theCHO cell supernatant. The rapid availability of antibody produced in thesame host cell and cell culture process as the final cGMP manufacturingprocess has the potential to reduce the duration of process developmentprograms. Lonza has developed a generic method using pooledtransfectants grown in CDACF medium, for the rapid production of smallquantities (up to 50 g) of antibodies in CHO cells. Although slightlyslower than a true transient system, the advantages include a higherproduct concentration and use of the same host and process as theproduction cell line. Example of growth and productivity of GS-CHOpools, expressing a model antibody, in a disposable bioreactor: in adisposable bag bioreactor culture (5 L working volume) operated infed-batch mode, a harvest antibody concentration of 2 g/L was achievedwithin 9 weeks of transfection.

pCon Vectors™ are an easy way to re-express whole antibodies. Theconstant region vectors are a set of vectors offering a range ofimmunoglobulin constant region vectors cloned into the pEE vectors.These vectors offer easy construction of full length antibodies withhuman constant regions and the convenience of the GS System™

Antibody molecules will comprise fragments (such as F(ab′), F(ab′)2)that are produced, for example, by the proteolytic cleavage of the mAbs,or single-chain immunoglobulins producible, for example, via recombinantmeans. Such antibody derivatives are monovalent. In one embodiment, suchfragments can be combined with one another, or with other antibodyfragments or receptor ligands to form “chimeric” binding molecules.Significantly, such chimeric

molecules may contain substituents capable of binding to differentepitopes of the same molecule.

It may be desirable to “humanize” antibodies produced in non-human hostsin order to attenuate any immune reaction when used in human therapy.Such humanized antibodies may be studied in an in vitro or an in vivocontext. Humanized antibodies may be produced, for example by replacingan immunogenic portion of an antibody with a corresponding, butnonimmunogenic portion (i.e., chimeric antibodies). PCT ApplicationPCT/US86/02269; EP Application 184,187; EP Application 171,496; EPApplication 173,494; PCT Application WO 20 86/01533; EP Application125,023; Sun et al. (1987); Wood et al. (1985); and Shaw et al. (1988);all of which references are incorporated herein by reference. Generalreviews of “humanized” chimeric antibodies are provided by Morrison(1985); also incorporated herein by reference. “Humanized” antibodiescan alternatively be produced by CDR or CEA substitution.

Jones et al. (1986); Verhoeyen et al. (1988); Beidler et al. (1988); allof which are incorporated herein by reference. In related embodiments,the antibody is a derivative of the disclosed antibodies, e.g., anantibody comprising the CDR sequences identical to those in thedisclosed antibodies (e.g., a chimeric, humanized or CDR-graftedantibody). In yet a further embodiment, the antibody is a fully humanrecombinant antibody.

Alternatively, one may wish to make modifications, such as introducingconservative changes into an antibody molecule. In making such changes,the hydropathic index of amino acids may be considered. The importanceof the hydropathic amino acid index in conferring interactive biologicfunction on a protein is generally understood in the art (Kyte andDoolittle, 1982). It is accepted that the relative hydropathic characterof the amino acid contributes to the secondary structure of theresultant protein, which in turn defines the interaction of the proteinwith other molecules, for example, enzymes, substrates, receptors, DNA,antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: basic amino acids: arginine (+3.0), lysine (+3.0), andhistidine (−0.5); acidic amino acids: aspartate (+3.0±1), glutamate(+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionicamino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), andthreonine (−0.4), sulfur containing amino acids: cysteine (−1.0) andmethionine (−1.3); hydrophobic, nonaromatic amino acids: valine (−1.5),leucine (−1.8), isoleucine (−1.8), proline (−0.5±1), alanine (−0.5), andglycine (0); hydrophobic, aromatic amino acids: tryptophan (−3.4),phenylalanine (−2.5), and tyrosine (−2.3).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity and produce a biologically orimmunologically modified protein. In such changes, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those that are within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine; glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.

The present disclosure also contemplates isotype modification. Bymodifying the Fc region to have a different isotype, differentfunctionalities can be achieved. For example, changing to IgG4 canreduce immune effector functions associated with other isotypes.Modified antibodies may be made by any technique known to those of skillin the art, including expression through standard molecular biologicaltechniques, or the chemical synthesis of polypeptides. Methods forrecombinant expression are addressed elsewhere in this document.

D. Single Chain Antibodies

A Single Chain Variable Fragment (scFv) is a fusion of the variableregions of the heavy and light chains of immunoglobulins, linkedtogether with a short (usually serine, glycine) linker. This chimericmolecule retains the specificity of the original immunoglobulin, despiteremoval of the constant regions and the introduction of a linkerpeptide. This modification usually leaves the specificity unaltered.These molecules were created historically to facilitate phage displaywhere it is highly convenient to express the antigen binding domain as asingle peptide.

Alternatively, scFv can be created directly from subcloned heavy andlight chains derived from a hybridoma. Single chain variable fragmentslack the constant Fc region found in complete antibody molecules, andthus, the common binding sites (e.g., protein A/G) used to purifyantibodies. These fragments can often be purified/immobilized usingProtein L since Protein L interacts with the variable region of kappalight chains.

Flexible linkers generally are comprised of helix- and turn-promotingamino acid residues such as alaine, serine and glycine. However, otherresidues can function as well. Tang et al. (1996) used phage display asa means of rapidly selecting tailored linkers for single-chainantibodies (scFvs) from protein linker libraries. A random linkerlibrary was constructed in which the genes for the heavy and light chainvariable domains were linked by a segment encoding an 18-amino acidpolypeptide of variable composition. The scFv repertoire (approx. 5×10⁶different members) was displayed on filamentous phage and subjected toaffinity selection with hapten. The population of selected variantsexhibited significant increases in binding activity but retainedconsiderable sequence diversity. Screening 1054 individual variantssubsequently yielded a catalytically active scFv that was producedefficiently in soluble form. Sequence analysis revealed a conservedproline in the linker two residues after the VH C-terminus and anabundance of arginines and prolines at other positions as the onlycommon features of the selected tethers.

The recombinant antibodies of the present disclosure may also involvesequences or moieties that permit dimerization or multimerization of thereceptors. Such sequences include those derived from IgA, which permitformation of multimers in conjunction with the J-chain. Anothermultimerization domain is the Gal4 dimerization domain. In otherembodiments, the chains may be modified with agents such asbiotin/avidin, which permit the combination of two antibodies.

In a separate embodiment, a single-chain antibody can be created byjoining receptor light and heavy chains using a non-peptide linker orchemical unit. Generally, the light and heavy chains will be produced indistinct cells, purified, and subsequently linked together in anappropriate fashion (i.e., the N-terminus of the heavy chain beingattached to the C-terminus of the light chain via an appropriatechemical bridge).

Cross-linking reagents are used to form molecular bridges that tiefunctional groups of two different molecules, e.g., a stablizing andcoagulating agent. However, it is contemplated that dimers or multimersof the same analog or heteromeric complexes comprised of differentanalogs can be created. To link two different compounds in a step-wisemanner, heterobifunctional cross-linkers can be used that eliminateunwanted homopolymer formation.

An exemplary hetero-bifunctional cross-linker contains two reactivegroups: one reacting with primary amine group (e.g., N-hydroxysuccinimide) and the other reacting with a thiol group (e.g., pyridyldisulfide, maleimides, halogens, etc.). Through the primary aminereactive group, the cross-linker may react with the lysine residue(s) ofone protein (e.g., the selected antibody or fragment) and through thethiol reactive group, the cross-linker, already tied up to the firstprotein, reacts with the cysteine residue (free sulfhydryl group) of theother protein (e.g., the selective agent).

It is preferred that a cross-linker having reasonable stability in bloodwill be employed. Numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate targeting andtherapeutic/preventative agents. Linkers that contain a disulfide bondthat is sterically hindered may prove to give greater stability in vivo,preventing release of the targeting peptide prior to reaching the siteof action. These linkers are thus one group of linking agents.

Another cross-linking reagent is SMPT, which is a bifunctionalcross-linker containing a disulfide bond that is “sterically hindered”by an adjacent benzene ring and methyl groups. It is believed thatsteric hindrance of the disulfide bond serves a function of protectingthe bond from attack by thiolate anions such as glutathione which can bepresent in tissues and blood, and thereby help in preventing decouplingof the conjugate prior to the delivery of the attached agent to thetarget site.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes thehetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxysuccinimidyl group reacts withprimary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use of suchcross-linkers is well understood in the art. Another embodiment involvesthe use of flexible linkers.

U.S. Pat. No. 4,680,338, describes bifunctional linkers useful forproducing conjugates of ligands with amine-containing polymers and/orproteins, especially for forming antibody conjugates with chelators,drugs, enzymes, detectable labels and the like. U.S. Pat. Nos. 5,141,648and 5,563,250 disclose cleavable conjugates containing a labile bondthat is cleavable under a variety of mild conditions. This linker isparticularly useful in that the agent of interest may be bonded directlyto the linker, with cleavage resulting in release of the active agent.Particular uses include adding a free amino or free sulfhydryl group toa protein, such as an antibody, or a drug.

U.S. Pat. No. 5,856,456 provides peptide linkers for use in connectingpolypeptide constituents to make fusion proteins, e.g., single chainantibodies. The linker is up to about 50 amino acids in length, containsat least one occurrence of a charged amino acid (preferably arginine orlysine) followed by a proline, and is characterized by greater stabilityand reduced aggregation. U.S. Pat. No. 5,880,270 disclosesaminooxy-containing linkers useful in a variety of

immunodiagnostic and separative techniques.

E. Purification

In certain embodiments, the antibodies of the present disclosure may bepurified. The term “purified,” as used herein, is intended to refer to acomposition, isolatable from other components, wherein the protein ispurified to any degree relative to its naturally-obtainable state. Apurified protein therefore also refers to a protein, free from theenvironment in which it may naturally occur. Where the term“substantially purified” is used, this designation will refer to acomposition in which the protein or peptide forms the major component ofthe composition, such as constituting about 50%, about 60%, about 70%,about 80%, about 90%, about 95% or more of the proteins in thecomposition.

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. Other methods for protein purification include,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; gel filtration, reversephase, hydroxylapatite and affinity chromatography; and combinations ofsuch and other techniques. In purifying an antibody of the presentdisclosure, it may be desirable to express the polypeptide in aprokaryotic or eukaryotic expression system and extract the proteinusing denaturing conditions. The polypeptide may be purified from othercellular components using an affinity column, which binds to a taggedportion of the polypeptide. As is generally known in the art, it isbelieved that the order of conducting the various purification steps maybe changed, or that certain steps may be omitted, and still result in asuitable method for the preparation of a substantially purified proteinor peptide.

Commonly, complete antibodies are fractionated utilizing agents (i.e.,protein A) that bind the Fc portion of the antibody. Alternatively,antigens may be used to simultaneously purify and select appropriateantibodies. Such methods often utilize the selection agent bound to asupport, such as a column, filter or bead. The antibodies is bound to asupport, contaminants removed (e.g., washed away), and the antibodiesreleased by applying conditions (salt, heat, etc.). Various methods forquantifying the degree of purification of the protein or peptide will beknown to those of skill in the art in light of the present disclosure.These include, for example, determining the specific activity of anactive fraction, or assessing the amount of polypeptides within afraction by SDS/PAGE analysis. Another method for assessing the purityof a fraction is to calculate the specific activity of the fraction, tocompare it to the specific activity of the initial extract, and to thuscalculate the degree of purity. The actual units used to represent theamount of activity will, of course, be dependent upon the particularassay technique chosen to follow the purification and whether or not theexpressed protein or peptide exhibits a detectable activity.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

III. PHARMACEUTICAL FORMULATIONS AND TREATMENT OF CANCER

A. Cancers

Cancer results from the outgrowth of a clonal population of cells fromtissue. The development of cancer, referred to as carcinogenesis. Unlikemany antibody therapies for cancer, the antibodies of the presentdisclosure are directed instead to the DC-HIL molecules found on thesurface of myeloid-derived suppressor cells (MDSCs).

Cancer cells to which the methods of the present disclosure can beapplied include generally any cancer that is subject to the influence ofMDSCs. An appropriate cancer cell can be a breast cancer, lung cancer,colon cancer, pancreatic cancer, renal cancer, stomach cancer, livercancer, bone cancer, hematological cancer (e.g., leukemia or lymphoma),neural tissue cancer, melanoma, ovarian cancer, testicular cancer,prostate cancer, cervical cancer, vaginal cancer, or bladder cancercell. In addition, the methods of the disclosure can be applied to awide range of species, e.g., humans, non-human primates (e.g., monkeys,baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs,cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Cancersmay also be recurrent, metastatic and/or multi-drug resistant, and themethods of the present disclosure may be particularly applied to suchcancers so as to render them resectable, to prolong or re-induceremission, to prevent or limit metastasis, and/or to treat multi-drugresistant cancers.

B. Formulation and Administration

The present disclosure provides pharmaceutical compositions comprisinganti-DC-HIL antibodies. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia or

other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

The term “carrier” refers to a diluent, excipient, or vehicle with whichthe therapeutic is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Other suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, saline, dextrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene glycol, water, ethanol and the like.

The compositions can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with anions suchas those derived from hydrochloric, phosphoric, acetic, oxalic, tartaricacids, etc., and those formed with cations such as those derived fromsodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

C. Combination Therapies

It may also be desirable to provide combination treatments usingantibodies of the present disclosure in conjunction with othertherapeutic modalities. These therapies would be provided in a combinedamount effective to achieve a reduction in one or more diseaseparameter. This process may involve contacting the cells/subjects withthe both agents/therapies at the same time, e.g., using a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell/subject with two distinct compositions orformulations, at the same time, wherein one composition includes theantibody and the other includes the other agent/therapy.

Alternatively, the antibody may precede or follow the other treatment byintervals ranging from minutes to weeks. One would generally ensure thata significant period of time did not expire between the time of eachdelivery, such that the therapies would still be able to exert anadvantageously combined effect on the cell/subject. In such instances,it is contemplated that one would contact the cell with both modalitieswithin about 12-24 hours of each other, within about 6-12 hours of eachother, or with a delay time of only about 12 hours. In some situations,it may be desirable to extend the time period for treatmentsignificantly; however, where several 10 days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

It also is conceivable that more than one administration of either theanti-DC-HIL antibody or the other therapy will be desired. Variouscombinations may be employed, where the antibody is “A,” and the othertherapy is “B,” as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are contemplated. To kill cells, inhibit cell growth,inhibit metastasis, inhibit angiogenesis or otherwise reverse or reducethe malignant phenotype of tumor cells, using the methods andcompositions of the present disclosure, one may contact a target cell orsite with an antibody and at least one other therapy. These therapieswould be provided in a combined amount effective to kill or inhibitproliferation of cancer cells. This process may involve contacting thecells/site/subject with the agents/therapies at the same time.

The antibodies of the disclosure may be particularly in enhancing theefficacy of cancer immunotherapies and vaccinations, anti-CTLA-4therapy, anti-PD-1 therapy, or radiation therapy.

D. Non-Antibody Cancer Therapies

As an adjunct to the diagnostic aspects of the present disclosure, itmay be desirable to make a treatment decision based on the outcome of adiagnostic method described herein, or to effect such a treatment.Treatment options are well known to those of skill in the art, and mayinclude the DC-HIL antibodies of the disclosure, as well asco-therapies. These are discussed above. However, it may also proveappropriate to treat patients with other “standard” therapies whereDC-HIL is not a relevant target. Again, in such situations, the goal maybe to kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells. Agents or factors suitable for cancer therapy include anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, irradiation, microwaves, electronicemissions, and the like. A variety of chemical compounds, also describedas “chemotherapeutic” or “genotoxic agents,” may be used. This may beachieved by irradiating the localized tumor site; alternatively, thetumor cells may be contacted with the agent by administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition.

Various classes of chemotherapeutic agents are comtemplated for use withthe present disclosure. For example, selective estrogen receptorantagonists (“SERMs”), such as Tamoxifen, 4-hydroxy Tamoxifen(Afimoxfene), Falsodex, Raloxifene, Bazedoxifene, Clomifene, Femarelle,Lasofoxifene, Ormeloxifene, and Toremifene. Chemotherapeutic agentscontemplated to be of use, include, e.g., camptothecin, actinomycin-D,or mitomycin C. The disclosure also encompasses the use of a combinationof one or more DNA damaging agents, whether radiation-based or actualcompounds, such as the use of X-rays with cisplatin or the use ofcisplatin with etoposide. The agent may be prepared and used as acombined therapeutic composition, or kit.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m2at 21 day intervals for doxorubicin, to 35-50 mg/m2 for etoposideintravenously or double the intravenous dose orally.

Microtubule inhibitors, such as taxanes, also are contemplated. Thesemolecules are diterpenes produced by the plants of the genus Taxus, andinclude paclitaxel and docetaxel. Epidermal growth factor receptorinhibitors, such as Iressa, mTOR, the mammalian target of rapamycin,also known as FK506-binding protein 12-rapamycin associated protein 1(FRAP1) is a serine/threonine protein kinase that regulates cell growth,cell proliferation, cell motility, cell survival, protein synthesis, andtranscription. Rapamycin and analogs thereof (“rapalogs”) are thereforecontemplated for use in cancer therapy in accordance with the presentdisclosure.

Another possible therapy is TNF-α (tumor necrosis factor-alpha), acytokine involved in systemic inflammation and a member of a group ofcytokines that stimulate the acute phase reaction. The primary role ofTNF is in the regulation of immune cells. TNF is also able to induceapoptotic cell death, to induce inflammation, and to inhibittumorigenesis and viral replication.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, x-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV irradiation. Itis most likely that all of these factors effect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for x-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

In addition, it also is contemplated that immunotherapy, hormonetherapy, toxin therapy and surgery can be used. It also should bepointed out that any of the foregoing therapies may prove useful bythemselves in treating cancer. The skilled artisan is directed to“Remington's Pharmaceutical Sciences” 15th Edition, Chapter 33, inparticular pages 624-652. Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

IV. ANTIBODY REAGENTS/CONJUGATES

Antibodies may be linked to at least one agent to form an antibodyconjugate. In order to increase the efficacy of antibody molecules asdiagnostic or therapeutic agents, it is conventional to link orcovalently bind or complex at least one desired molecule or moiety. Sucha molecule or moiety may be, but is not limited to, at least oneeffector or reporter molecule. Effector molecules comprise moleculeshaving a desired activity, e.g., immunosuppression/anti-inflammation.Such molecules are optionally attached via cleavable linkers designed toallow the molecules to be released at or near the target site.Non-limiting examples of reporter molecules which have been conjugatedto antibodies include enzymes, radiolabels, haptens, fluorescent labels,phosphorescent molecules, chemiluminescent molecules, chromophores,photoaffinity molecules, colored particles or ligands, such as biotin.

Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and those for use in vivo diagnostic protocols, generally known as“antibody-directed imaging.” Many appropriate imaging agents are knownin the art, as are methods for their attachment to antibodies (see, fore.g., U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509). The imagingmoieties used can be paramagnetic ions, radioactive isotopes,fluorochromes, NMR-detectable substances, and X-ray imaging agents. Inthe case of paramagnetic ions, one might mention by way of example ionssuch as chromium (III), manganese (II), iron (III), iron (II), cobalt(II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹4carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium⁹⁹m and/oryttrium⁹⁰. ¹²⁵I is often being preferred for use in certain embodiments,and technicium⁹⁹m and/or indium¹¹¹ are also often preferred due to theirlow energy and suitability for long range detection. Radioactivelylabeled monoclonal antibodies may be produced according to well-knownmethods in the art. For instance, monoclonal antibodies can be iodinatedby contact with sodium and/or potassium iodide and a chemical oxidizingagent such as sodium hypochlorite, or an enzymatic oxidizing agent, suchas lactoperoxidase. Monoclonal antibodies may be labeled withtechnetium⁹⁹m by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column.Alternatively, direct labeling techniques may be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the antibody.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of antibody conjugates contemplated are those intendedprimarily for use in vitro, where the antibody is linked to a secondarybinding ligand and/or to an enzyme (an enzyme tag) that will generate acolored product upon contact with a chromogenic substrate. Examples ofsuitable enzymes include urease, alkaline phosphatase, (horseradish)hydrogen peroxidase or glucose oxidase. Preferred secondary bindingligands are biotin and avidin and streptavidin compounds. The use ofsuch labels is well known to those of skill in the art and described,for example, in U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350,3,996,345, 4,277,437, 4,275,149 and 4,366,241.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter and Haley, 1983).In particular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; Dholakia et al., 1989) and may be used as antibodybinding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may alsobe reacted with an enzyme in the presence of a coupling agent such asglutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breast tumors isachieved using monoclonal antibodies and the detectable imaging moietiesare bound to the antibody using linkers such asmethyl-phydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

V. IMMUNODETECTION METHODS

In still further embodiments, there are immunodetection methods forbinding, purifying, removing, quantifying and otherwise generallydetecting DC-HIL and its associated antigens. Some immunodetectionmethods include enzyme linked immunosorbent assay (ELISA),radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay,chemiluminescent assay, bioluminescent assay, and Western blot tomention a few. In particular, a competitive assay for the detection andquantitation of DC-HIL antibodies also is provided. The steps of varioususeful immunodetection methods have been described in the scientificliterature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis andGaland (1993), De Jager et al. (1993), and Nakamura et al. (1987).

In general, the immunobinding methods include obtaining a sample andcontacting the sample with a first antibody in accordance withembodiments discussed herein, as the case may be, under conditionseffective to allow the formation of immunocomplexes. Contacting thechosen biological sample with the antibody under effective conditionsand for a period of time sufficient to allow the formation of immunecomplexes (primary immune complexes) is generally a matter of simplyadding the antibody composition to the sample and incubating the mixturefor a period of time long enough for the antibodies to form immunecomplexes with, i.e., to bind to DC-HIL present. After this time, thesample-antibody composition, such as a tissue section, ELISA plate, dotblot or Western blot, will generally be washed to remove anynon-specifically bound antibody species, allowing only those antibodiesspecifically bound within the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. Patents concerning the use of such labelsinclude U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345,4,277,437, 4,275,149 and 4,366,241. Of course, one may find additionaladvantages through the use of a secondary binding ligand such as asecond antibody and/or a biotin/avidin ligand binding arrangement, as isknown in the art.

The antibody employed in the detection may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo-step approach. A second binding ligand, such as an antibody that hasbinding affinity for the antibody, is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection uses two different antibodies. A firstbiotinylated antibody is used to detect the target antigen, and a secondantibody is then used to detect the biotin attached to the complexedbiotin. In that method, the sample to be tested is first incubated in asolution containing the first step antibody. If the target antigen ispresent, some of the antibody binds to the antigen to form abiotinylated antibody/antigen complex. The antibody/antigen complex isthen amplified by incubation in successive solutions of streptavidin (oravidin), biotinylated DNA, and/or complementary biotinylated DNA, witheach step adding additional biotin sites to the antibody/antigencomplex. The amplification steps are repeated until a suitable level ofamplification is achieved, at which point the sample is incubated in asolution containing the second step antibody against biotin. This secondstep antibody is labeled, as for example with an enzyme that can be usedto detect the presence of the antibody/antigen complex byhistoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

A. ELISAs

Immunoassays, in their most simple and direct sense, are binding assays.Certain preferred immunoassays are the various types of enzyme linkedimmunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in theart. Immunohistochemical detection using tissue sections is alsoparticularly useful. However, it will be readily appreciated thatdetection is not limited to such techniques, and western blotting, dotblotting, FACS analyses, and the like may also be used.

In one exemplary ELISA, the antibodies of the disclosure are immobilizedonto a selected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a test composition suspected ofcontaining the DC-HIL is added to the wells. After binding and washingto remove non-specifically bound immune complexes, the bound antigen maybe detected.

Detection may be achieved by the addition of another anti-DC-HILantibody that is linked to a detectable label. This type of ELISA is asimple “sandwich ELISA.” Detection may also be achieved by the additionof a second anti-DC-HIL antibody, followed by the addition of a thirdantibody that has binding affinity for the second antibody, with thethird antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing theDC-HIL antigen are immobilized onto the well surface and then contactedwith anti-DC-HIL antibody. After binding and washing to removenon-specifically bound immune complexes, the bound anti-DC-HILantibodies are detected. Where the initial anti-DC-HIL antibodies arelinked to a detectable label, the immune complexes may be detecteddirectly. Again, the immune complexes may be detected using a secondantibody that has binding affinity for the first anti-DC-HIL antibody,with the second antibody being linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below. In coating a plate with eitherantigen or antibody, one will generally incubate the wells of the platewith a solution of the antigen or antibody, either overnight or for aspecified period of hours. The wells of the plate will then be washed toremove incompletely adsorbed material. Any remaining available surfacesof the wells are then “coated” with a nonspecific protein that isantigenically neutral with regard to the test antisera. These includebovine serum albumin (BSA), casein or solutions of milk powder. Thecoating allows for blocking of nonspecific adsorption sites on theimmobilizing surface and thus reduces the background caused bynonspecific binding of antisera onto the surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween). Afterincubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic 5 substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H2O2, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

B. Western Blot

The Western blot (alternatively, protein immunoblot) is an analyticaltechnique used to detect specific proteins in a given sample of tissuehomogenate or extract. It uses gel electrophoresis to separate native ordenatured proteins by the length of the polypeptide (denaturingconditions) or by the 3-D structure of the protein(native/non-denaturing conditions). The proteins are then transferred toa membrane (typically nitrocellulose or PVDF), where they are probed(detected) using antibodies specific to the target protein.

Samples may be taken from whole tissue or from cell culture. In mostcases, solid tissues are first broken down mechanically using a blender(for larger sample volumes), using a homogenizer (smaller volumes), orby sonication. Cells may also be broken open by one of the abovemechanical methods. However, it should be noted that bacteria, virus orenvironmental samples can be the source of protein and thus Westernblotting is not restricted to cellular studies only. Assorteddetergents, salts, and buffers may be employed to encourage lysis ofcells and to solubilize proteins. Protease and phosphatase inhibitorsare often added to prevent the digestion of the sample by its ownenzymes. Tissue preparation is often done at cold temperatures to avoidprotein denaturing.

The proteins of the sample are separated using gel electrophoresis.Separation of proteins may be by isoelectric point (pI), molecularweight, electric charge, or a combination of these factors. The natureof the separation depends on the treatment of the sample and the natureof the gel. This is a very useful way to determine a protein. It is alsopossible to use a two-dimensional (2-D) gel which spreads the proteinsfrom a single sample out in two dimensions. Proteins are separatedaccording to isoelectric point (pH at which they have neutral netcharge) in the first dimension, and according to their molecular weightin the second dimension.

In order to make the proteins accessible to antibody detection, they aremoved from within the gel onto a membrane made of nitrocellulose orpolyvinylidene difluoride (PVDF). The membrane is placed on top of thegel, and a stack of filter papers placed on top of that. The entirestack is placed in a buffer solution which moves up the paper bycapillary action, bringing the proteins with it. Another method fortransferring the proteins is called electroblotting and uses an electriccurrent to pull proteins from the gel into the PVDF or nitrocellulosemembrane. The proteins move from within the gel onto the membrane whilemaintaining the organization they had within the gel. As a result ofthis blotting process, the proteins are exposed on a thin surface layerfor detection (see below). Both varieties of membrane are chosen fortheir non-specific protein binding properties (i.e., binds all proteinsequally well). Protein binding is based upon hydrophobic interactions,as well as charged interactions between the membrane and protein.Nitrocellulose membranes are cheaper than PVDF, but are far more fragileand do not stand up well to repeated probings. The uniformity andoverall effectiveness of transfer of protein from the gel to themembrane can be checked by staining the membrane with CoomassieBrilliant Blue or Ponceau S dyes. Once transferred, proteins aredetected using labeled primary antibodies, or unlabeled primaryantibodies followed by indirect detection using labeled protein A orsecondary labeled antibodies binding to the Fc region of the primaryantibodies.

C. Immunohistochemistry

The antibodies may also be used in conjunction with both fresh-frozenand/or formalinfixed, paraffin-embedded tissue blocks prepared for studyby immunohistochemistry (IHC). The method of preparing tissue blocksfrom these particulate specimens has been successfully used in previousIHC studies of various prognostic factors, and is well known to those ofskill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred etal., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections fromthe capsule. Alternatively, whole frozen tissue samples may be used forserial section cuttings. Permanent-sections may be prepared by a similarmethod involving rehydration of the 50 mg sample in a plastic microfugetube; pelleting; resuspending in 10% formalin for 4 hours fixation;washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling inice water to harden the agar; removing the tissue/agar block from thetube; infiltrating and/or embedding the block in paraffin; and/orcutting up to 50 serial permanent sections. Again, whole tissue samplesmay be substituted.

D. Immunodetection Kits

In still further embodiments, there are immunodetection kits for usewith the immunodetection methods described above. The immunodetectionkits will thus comprise, in suitable container means, a first antibodythat binds to DC-HIL antigen, and optionally an immunodetection reagent.

In certain embodiments, the DC-HIL antibody may be pre-bound to a solidsupport, such as a column matrix and/or well of a microtitre plate. Theimmunodetection reagents of the kit may take any one of a variety offorms, including those detectable labels that are associated with orlinked to the given antibody. Detectable labels that are associated withor attached to a secondary binding ligand are also contemplated.Exemplary secondary ligands are those secondary antibodies that havebinding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody, along with a thirdantibody that has binding affinity for the second antibody, the thirdantibody being linked to a detectable label. As noted above, a number ofexemplary labels are known in the art and all such labels may beemployed in connection with embodiments discussed herein.

The kits may further comprise a suitably aliquoted composition of theDC-HIL antigen, whether labeled or unlabeled, as may be used to preparea standard curve for a detection assay. The kits may containantibody-label conjugates either in fully conjugated form, in the formof intermediates, or as separate moieties to be conjugated by the userof the kit. The components of the kits may be packaged either in aqueousmedia or in lyophilized form.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the antibody may be placed, or preferably, suitably aliquoted. Thekits will also include a means for containing the antibody, antigen, andany other reagent containers in close confinement for commercial sale.Such containers may include injection or blow-molded plastic containersinto which the desired vials are retained.

VI. EXAMPLES

The following examples are included to demonstrate preferredembodiments. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof embodiments, and thus can be considered to constitute preferred modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of thedisclosure.

Example 1—Materials and Methods

Reagents. Ab against CD3 (145-2C11), CD11b (M1/70), CD11c (N418), CD14(61D3), CD19 (eBio1D3), CD28 (37,51), CD80 (16A-10A1), CD86 (GL1), Gr-1(RB6-8C5), HLA-DR (LN3), IFN-γ (XMG1.2), IL-10 (JES5-16E3), PD-L1(MIH5), TGF-β1 (9016), Thy1.1 (HIS51) and control Ab were purchased fromeBioscience; anti-phosphotyrosine (4G10) from Upstate Biotechnology; andall recombinant cytokines from Pepro Tech. The inventors generated 1E4rat anti-mouse DC-HIL and UTX103 rabbit anti-mouse DC-HIL as describedpreviously (Chung et al., 2009). DC-HIL-Fc fusion protein was producedby COS-1 cells and purified (Chung et al., 2007). The chemicalinhibitors, L-NG-monomethyl-arginine citrate,N6-(1-iminoethyl)-L-lysine, N-hydroxyl-nor-arginine,1-methyl-tryptophan, and catalase and superoxide dismutase, werepurchased from Sigma-Aldrich. hgp100 peptide (KVPRNQDWL), OVA257-264H-2Kb-class I (SIINFEKL), and OVA323-339 H-2Kb-class II peptide(ISQAVHAAHAEINEAGR) were synthesized by the Protein Chemistry TechnologyCenter at UT Southwestern.

Animals and Cell Culture.

Female C57BL/6 mice and pmel-1 TCR transgenic mice(B6.Cg-Thy1a/CyTg(TcraTcrb)8Rest/J) (5- to 8-wk-old) were purchased fromHarlan Breeders and Jackson Laboratory, respectively. The inventorsgenerated DC-HIL gene-disrupted C57BL/6 mice: The left arm (5.1 Kb) andright arm (3.1 Kb) were isolated from a mouse chromosome BAC clone (fromC57BL/6 mice) and inserted into the 5′-end and 3′-end, respectively, ofthe neo gene in pVPTK01 targeting vector. This vector was linearized bydigestion of restriction enzyme I-CeuI (New England Biolabs) andelectroporated into C57BL/6-derived embryonic stem cells (inGenioustargeting laboratories). The targeted DC-HIL mutation-bearing ES cellswere screened by differential sensitivity to G418 and TK and Southernblotting of genome DNA. Chimeric mice were produced by microinjection ofblastocysts and backcrossed with C57BL/6 for 6 generations, and KO miceproduced by breeding heterozygotes. SD-4-deficient mice (C57BL/6 geneticbackground) were obtained from by Dr. Kojima (Nagoya University)(Ishiguro et al., 2000). Control groups included mice with WT genotype(DC-HIL^(+/+) or SD-4^(+/+)) from the same generation of backcross.SD-4-deficient pmel-1 mice were produced by breeding SD-4-deficient miceand pmel-1 transgenic mice. Following NIH guidelines, animals werehoused in the pathogen-free facility of the Institutional Animal CareUse Center of The University of Texas Southwestern Medical Center. Allanimal protocols were approved by the Center. B16F10 (B16) melanomacells were purchased from the American Type Culture Collection andmaintained in DMEM supplemented with 10% FCS. KD-B16 melanoma cells weregenerated (28). Freshly isolated leukocytes were cultured in 10%FCS-RPMI 1640.

Preparation of Leukocytes.

For MDSC, B16F10 melanoma cells (5×10⁵) were harvested by treatment with5% EDTA/trypsin, washed, resuspended in 50 μl of DPBS, and then injecteds.c. into the right shaved flank of WT or KO mice (3-10 mice perexperiment). Three weeks later, spleen cells were pooled from mice,depleted of CD11c+ and CD19+ cells using 10 μl of anti-biotin-beads(Invitrogen) coated with corresponding Ab, incubated with anti-CD11bAb-coated magnetic beads (Miltenyi Biotec), and then applied to amagnetic column. Eluate is the CD11b+ cell fraction (MDSC preparation)and the pass through is the CD11b^(neg) fraction (control cells forMDSC). Normally, MDSC preparations contain ˜95% CD11b+Gr-1+ cells. MDSCwere also isolated from BM and peripheral blood using the same method.Tumor-infiltrating cells were prepared from B16 tumor (˜2 cm³): Thetumor was minced in PBS, treated with digestive enzyme mixture [0.5mg/ml Collagenase I and IV and 0.1 mg/ml DNase I (Sigma-Aldrich)] at 37°C. for 1 h, and applied to Ficoll-gradient to remove cell debris. BM-DCwere harvested from day 6 culture of BM cells isolated from C57BL/6 mice(Chung et al., 2009). Macrophages and DC were also purified from spleencells prepared from mice bearing B16 tumor (3 weeks after tumorinoculation) by FACS sorting of F4/80+ and CD11c+ cells, respectively.These preparations contain ˜95% of macrophage and DC, respectively.

Flow Cytometry (FACS).

Leukocytes (1-5×10⁵ cells) were incubated with primary mAb or theisotypic control IgG (each 1-10 μg/ml), and labeled with fluorescentsecondary Ab (1 μg/ml). Fluorescence intensity of stained cells wasanalyzed by FACSCalibur (BD Biosciences).

Ag Presentation Assay.

BM-DC prepared from BM cells of KO or WT mice were seeded on 96well-plate (1-20×10³ cells/well), and pulsed for 3 hr with OVA peptide(2 μg/ml). Then, CD4+ or CD8+ T cells (1×10⁵/well) from OT-I or OT-IItransgenic mice, respectively were added to the culture. One day aftercoculture, IL-2 and/or IFN-γ in the culture supernatant were measured byELISA. To assay the T cell-stimulatory capacity of myeloid cellsisolated from mice with B16 tumor, purified DC, macrophages or MDSC(increasing cell ratios to T cell) were added to pmel-1 spleen cells(1×10⁵ cells/well) with 1 μg/ml hgp100 peptide. After culturing for 2 d,proliferative response was measured by ³H-thymidine (1 μCi/well)incorporation with pulsing in the last 20 h of the culture period. Netcpm in T cells is calculated by subtracting background cpm in culture ofmyeloid cell alone plus peptide from cpm in the coculture.

T Cell Suppression Assay.

MDSC were purified from spleen cells of mice with B16 tumor andcocultured with pmel-1 spleen cells (2×10⁵/well) at varying cell ratiosin the presence of 1 μg/ml hgp100 peptide for 3 d. T cell activation wasmeasured by ³H-thymidine incorporation. For inhibition studies,inhibitors (varying doses of Ab or chemicals) were added separately toculture of spleen cells/MDSC (1:1 cell ratio). To examine impact ofDC-HIL deletion on the suppressor activity, increasing doses of MDSCpurified from WT or DC-HIL KO mice bearing B16 tumor were coculturedwith pmel-1 spleen cells (2×10⁵/well). Alternatively, purified MDSC weredepleted of DC-HIL+ cells using 10 μl of anti-biotin-beads (Invitrogen)precoated with anti-DC-HIL mAb (or IgG as control). To examine impact ofSD-4 deletion on T cell susceptibility to MDSC function, spleen cells(2×10⁵/well) from SD-4+/+ or SD-4−/− pmel-1 transgenic mice werecocultured with 0.1 μg/ml hgp100 peptide and varying numbers ofmelanoma-MDSC.

To assay the ability of MDSC to suppress in vivo T cell response, day 0WT mice (n=5) were i.v. injected with pmel-1 CD8+ T cells (1×10⁷cells/mouse); day 3 they were also given i.v. injection of IgG-treatedor DC-HIL-depleted MDSC and vaccinated with complete Freund's adjuvantplus gp100 peptide (1 mg/ml); and day 13 draining inguinal LN wereprocured from all mice and cultured for 2 d in Ab-coated wells withgp100 peptide (1 μg/ml). Cells secreting IFN-γ were counted by ELISPOTassay (eBiosciences).

Tyrosine Phosphorylation Assay.

Purified MDSC (5×10⁶ cells) were treated with UTX103 mAb or controlrabbit IgG (10 μg/ml) on ice for 30 min, followed by crosslinking with100 μg/ml goat anti-rabbit IgG. At indicated time periods at 37° C.,treated MDSC were lysed using 500 μl of 2× lysis buffer (Chung et al.,2009). DC-HIL protein was immunoprecipitated by incubation at 4° C. for3 h with 2-5 jag of UTX103 mAb and 2 h incubation with protein-A agarose(50 μl of 50% slurry). Immune complexes were then analyzed forexpression of phosphotyrosine by immunoblotting using biotinylatedanti-phosphotyrosine (0.5 μg/ml) and HRP-streptavidin (1/10,000dilution) (Chung et al., 2009). Blotted membranes were stripped andreanalyzed using 1E4 rat anti-DC-HIL mAb (1 μg/ml) and HRP-anti-rat IgG(1/10,000 dilution). Image acquisition and analysis of Ab-reactive bandswere performed using ImageQuant 400 (Amersham Biosciences).

Tumor Growth and Metastasis Assays.

B16 cells (5×10⁵) were injected s.c. into the right flank of WT orDC-HIL KO mice (n=9). Tumor growth was monitored every other day bymeasuring perpendicular diameters using a metric caliper, and tumorvolume estimated (Kamijo et al., 1994). To examine the ability of MDSCto promote tumor growth, MDSC (2×10⁶) purified from melanoma-bearing KOor WT mice (n=10) were mixed with 2×10⁵ B16 cells in a total volume of50 μl DPBS, and then injected s.c. into naive WT mice (n=5). A weeklater, mice were i.v. injected again with purified MDSC (2×10⁶cells/mouse). Control mice were injected with B16F10 cells alone. Tumorvolume was monitored.

For lung metastasis, B16 cells (1×10⁶ cells) were harvested, resuspendedin 200 μl of DPBS, and injected into WT or DC-HIL KO mice (n=10) via thelateral tail veil. Lungs were harvested 18 or 19 d post-injection, andtheir total weight, number of metastatic foci, and melanin content weredetermined (Tomihari et al., 2010).

Assays for Soluble Factors.

Melanoma-MDSC were cultured in 96-well plates (2×10⁵ cells/well, intriplicate) precoated with UTX103 mAb or control IgG (10 μg/ml). After 1or 2 d of culture at 37° C., the culture supernatant and cell pelletswere collected separately: the former tested cytokine secretion (IFN-γ,TNF-α, IL-10, and TGF-β1) using ELISA; and the latter tested whole cellextracts (to measure NO production using Griess method (De Santo et al.,2005)) or total RNA (to measure IFN-γ and NOS mRNA using quantitativePCR). The inventors also measured soluble factors in MDSC purified fromspleen of WT or KO mice (n=5) with B16 tumor, which included NO,arginase activity (assayed by a chromogen that measures urea produced bythe enzyme) (Rodriguez et al., 2005), and ROS by DCFH-DA fluorescent dye(Kim et al., 2010).

Quantitative PCR.

IFN-γ and NOS mRNA expression in total RNA samples was assayed byquantitative PCR following the manufacturer's recommendations(LightCycler FastStart DNA Masterplus SYBR Green I, Roche). Primers forIFN-γ, 5′-AGTGGAGCAGGTGAAGAGTG-3′ (SEQ ID NO: 24) and5′-TTCGGAGAGAGGTACAAACG-3′ (SEQ ID NO: 25), for NOS-1,5′-TGTGCTTTGATGGAGATGAGG-3′(SEQ ID NO: 26) and5′-CAAAGTTGTCTCTGAGGTCTGG-3′ (SEQ ID NO: 27), for NOS-2 are5′-AGAGTGAAAAGTCCAGCCG-3′ (SEQ ID NO: 28) and 5′-ACAACTCGCTCCAAGATTCC-3′(SEQ ID NO: 29), and for NOS-3 are 5′-CTGCCACCTGATCCTAACTTG-3′ (SEQ IDNO: 30) and 5′-CAGCCAAACACCAAAGTCATG-3′ (SEQ ID NO: 31). PCRamplification efficiencies were determined for each gene prior to therelative quantification and were similar for the target gene and theendogenous control (GAPDH). mRNA expression for each sample wasexpressed as the expression level relative to GAPDH gene, which wasquantitated using the comparative Ct method and the formula 2-ΔΔCT.

Ab Treatment of Mice.

On day 0, C57BL/6 (14 mice) were inoculated s.c. with 5×105 B16 orKD-B16 cells (Tomihari et al., 2010). On day 6 (for mice bearing B16F10tumor) or day 11 (for KD-B16 tumor), mice were sorted to 2 groups withsimilar average tumor volume (˜0.1 cm3) and i.p. injected with UTX103mAb or rabbit IgG (200 μg/mouse) every other day for a total of 5-6injections.

To examine the effect of UTX103 mAb on T cell-stimulatory capacity ofDC, DC-HIL^(−/−) mice (12 mice) were implanted s.c. with 5×10⁵ B16cells, and 2 weeks later mice were sorted into 3 groups (n=4) withsimilar average tumor volume (˜0.2 cm³). The same day (day 0), the micewere i.v. injected with 1×10⁷ CFSE-labeled pmel-1 T cells andimmediately afterwards i.v. injected with 5×10⁶ BM-DC unpulsed or pulsedfor 2 h with the corresponding Ag (1 μg/ml). On days 0 and 2, UTX103 mAbor control IgG (200 μg/mouse) was injected i.p. into treated mice. Onday 3, spleen and draining LN were procured and assayed for CFSEfluorescence intensity of Thy1.1+ cells by FACS.

Circulating MDSC.

At indicated time points before or after Ab injection into mice bearingmelanoma, 5 representative mice for each group were chosen; bloodsamples (50-100 μl) were taken from mouse tails, depleted of red bloodcells by lysis, pulsed with OVA257-264 peptide (1 μg/ml), and stainedwith PE anti-OVA-H-2Kb (to count nucleated cells), APC anti-CD11b, andFITC anti-Gr-1 for FACS analysis. H-2Kb+ cells were gated and examinedfor % of CD11b+Gr-1+ MDSC.

Melanoma Patients and Controls.

The study was approved by The University of Texas Southwestern MedicalCenter Institutional Review Board and was conducted according toprinciples of the Declaration of Helsinki. Participants gave writteninformed consent in accordance with the Declaration of Helsinki. Bloodsamples were taken from 6 healthy donors and 6 melanoma patients. Allpatients were classified as stage III metastatic melanoma (AmericanJoint Committee on Cancer criteria) and had been treated previously withimmunotherapy. Six patients had a median age of 53 years and included 4women and 2 men.

Human MDSC and T Cell Assay.

PBMCs (5×10⁵ cells/reaction) isolated using cell preparation tube withsodium citrate (BD Vacutainer CPT, BD Biosciences) were treated withhuman IgG and incubated with 10 μg/ml 3D5 anti-human DC-HIL mAb (orcontrol IgG) and 1 μg/ml PE-anti-mouse IgG [F(ab′)₂ fragment]. Afterwashing, cells were further stained with APC anti-HLA-DR and FITCanti-CD14 Ab (each 10 μg/ml), and analyzed by FACS.

CD14⁺HLA-DR^(no/low) MDSC, HLA-DR+ cells, and T cells were freshlyisolated from blood samples of same donor: PBMCs from a melanoma patient(or healthy donors) were fractionated into HLA-DR^(neg) and HLA-DR+cells (control) using magnetic beads (Miltenyi Biotec). The formerfraction was incubated with anti-CD14 Ab-coated magnetic beads(Miltenyi), and then applied to a column. The column eluate (the MDSCfraction containing >95% of CD14⁺HLA-DR^(no/low) cells), and thepass-through fraction was used for isolation of T cells using Pan-T cellisolation kit (Miltenyi). MDSC or HLA-DR+ control cells were coculturedwith autologous T cells (1×10⁵ cells/well) at varying cell ratios in thepresence of anti-CD2/CD3/CD28 beads (Miltenyi) (1.5 beads per T cell) in96 microculture wells (triplicate) for 5 d. For inhibition studies,varying doses of 3D5 mAb or control IgG were added to the same culture(1:1 cell ratio). IFN-γ production was determined by ELISA, andsuppressive activity of MDSC was assessed by IFN-γ amount (%) relativeto that of control culture (corresponding HLA-DR+/T cell culture).

Statistical Analysis.

Statistical analyses were performed using student's t test. In allcases, p values were calculated using two-sided t test, with p (<0.01)considered significant. All data shown are representative of at least 2independent experiments.

Example 2—Results

DC-HIL Gene Disruption Enhances the T Cell-Stimulatory Capacity of DC.

To study the in vivo significance of the DC-HIL receptor on APC, theinventors created DC-HIL gene-knocked out (KO) mice by replacing afragment spanning exons 2-4 with a targeting vector (FIG. 1A) to producea frame-shift replacement in downstream exons caused by mismatchedacceptor-donor sites for RNA splicing between exons 1 and 5. Thetargeted DC-HIL mutation was introduced into C57BL/6 mice, and the KOallele confirmed by PCR analysis of genomic DNA (FIG. 1B).

DC-HIL mRNA expression in KO mice was examined by RT-PCR analysis oftotal RNA isolated from BM-derived DC (BM-DC) using 2 primer sets: thefirst to amplify exons 2-3 (within targeted region), and the second forexons 5-9 (downstream) (FIG. 1C). The first primer amplified RNA from DCof WT mice, but not of KO mice; and the second primer produced PCRproduct from both RNA samples, indicating that KO mRNA lacked targetedexons but bore the remaining downstream 5-9 exons. The inventors thenprobed for DC-HIL protein using 2 mAb (FIG. 1C), UTX103 rabbitanti-DC-HIL and 1E4 rat anti-DC-HIL mAb, whose epitopes are encoded byexons 2 and 8, respectively. Both mAb stained 2 bands (95 and 120 KDa)from extracts of DC from WT mice (but not from KO mice) (FIG. 1D).Control β-actin expression was similar for both. Moreover, UTX103 mAbfailed to show surface expression of DC-HIL on KO-DC (FIG. 1E). Therewas no significant difference in the frequency of CD11c+ cells amongGM-CSF-cultured BM cells (91% for WT vs. 88% for KO). Similar resultswere shown for macrophages (FIG. 1F).

The inventors did not observe any gross abnormality or developmentaldefect in lymphoid organs of KO mice, and there was no significantdifference in the proportions of leukocyte subpopulations compared to WT(data not shown). The inventors then evaluated the capacity ofDC-HIL^(−/−) DC to activate syngeneic OVA-specific T cells (FIG. 1G).Increasing numbers of DC from WT or KO mice were cocultured with CD4+ orCD8+ T cells in the presence of OVA peptides. DC-HIL^(−/−) DC stimulatednaive CD4+ and CD8+ T cells to produce IL-2 and IFN-γ at 2-fold greaterlevels than DC-HIL^(+/+) DC. Since lack of DC-HIL had no impact onconstitutive expression of costimulatory receptors (CD80 and CD86, FIG.1H), these results confirm DC-HIL receptor to be a negative regulator onAPC.

Deletion of DC-HIL Gene in Mice Inhibits Growth of Subcutaneous andMetastatic Melanoma.

The inventors next employed DC-HIL^(−/−) mice to assess influence ofDC-HIL on melanoma growth. DC-HIL-KO or WT mice were inoculated s.c.with B16 melanoma cells (FIG. 2A). Growth of subcutaneous melanoma wasaggressive in WT mice, but markedly inhibited in KO mice (2.5±0.3 vs.0.6±0.1 cm3 on day 15). They then examined impact of DC-HIL deletion onlung metastasis (or spreading to lung) (FIGS. 2C-D). WT or KO mice wereinfused with B16 cells via the tail vein, and their lungs examined after19 days: Compared to WT mice, KO mice had markedly lighter lungs(0.28±0.06 g for WT vs. 0.15±0.02 g for KO), less metastatic foci(1,280±636 vs.194±105), less melanin content per lung (0.89±0.44 vs.0.045±0.021 mg), and less melanin per metastatic focus (0.7±0.1 vs.0.2±0.08 μg).

The inventors' previous finding that DC-HIL on B16 melanoma cellspromotes melanoma growth by suppressing T cell activation promptedexamination of the influence of melanoma-expressed DC-HIL on the tumorgrowth inhibition in DC-HIL KO mice. DC-HIL-knocked down B16 (KD-B16)cells were subcutaneously implanted into WT or KO mice, and tumor growthmeasured (FIG. 2B). KD-B16 cells grew in WT mice more slowly thanparental B16 cells (Tomihari et al., 2010), and this slow growth wasalso markedly inhibited in KO mice. These results indicate thathost-associated DC-HIL fuels aggressive tumor growth, independent ofmelanoma-associated DC-HIL.

Among DC-HIL+ Myeloid Cells in Mice Bearing Melanoma, CD11b+Gr-1+ Cellsare the Major Population Responsible for the Most Potent TCell-Suppressive Activity.

Because DC-HIL is expressed by APC or myeloid cells (but not bylymphocytes) in spleen of naive or immunized mice (Chung et al., 2009),the inventors wanted to know which DC-HIL+ myeloid cells exerted themost influence on melanoma growth. The inventors analyzed the phenotypeof spleen cells expressing DC-HIL in mice bearing B16 melanoma (FIGS.2A-B). Proportions of CD11c+ DC or F4/80+ macrophages were mildlyincreased (6-7%) compared with tumor-free mice (3-5%). Afterimplantation of B16 melanoma, the most dramatically expanded myeloidpopulation was CD11b+Gr-1+ cells (10-fold increase of 2% to 20%).Moreover, % of DC-HIL+ cells was markedly increased in all 3 populations(50-80%), but the CD11b+Gr-1+ phenotype was the major DC-HIL+ myeloidpopulation in spleen (˜10%). The inventors refer to this phenotype asmyeloid-derived suppressor cells (MDSC).

Since DC-HIL is a negative regulator of T cell activation, the inventorsexamined which DC-HIL+ myeloid subset is the most potent suppressor inmice bearing melanoma (FIG. 3D). Each myeloid population was purifiedfrom melanoma-bearing mice and cocultured with naive T cells activatedby anti-CD3/CD28 Ab, and T cell activation measured by CFSEproliferation assay. In the absence of myeloid cells, the costimulationcaused 61% of T cells to proliferate in 2 days. F4/80+ macrophages andCD11c+ DC were unable to inhibit such T cell activation even at a highcell ratio (1:1), whereas MDSC inhibited it in a dose-dependent mannerand with almost complete suppression at the highest ratio. Because DCand macrophages are considered APC, the inventors assayed their Tcell-stimulatory capacity in comparison to MDSC (FIG. 3D). Naive CD8+ Tcells from pmel-1 TCR transgenic mice (in which all CD8+ T cells expressthe same TCR specific to gp100 peptide (Overwijk et al. 2003)) weremixed with different myeloid cells pulsed with gp100 peptide. At a cellratio of 1:0.5, CD11c+ DC or F4/80+ macrophages induced strong T cellproliferation. Increasing the dose (1:0.5 and 1:1) did not furtheraugment T cell activation, indicating 1:0.25 as the peak dose. At thesame dose, MDSC also exhibited similar T cell-stimulatory capacity, butincreasing doses led to less proliferation to almost completesuppression. Thus, MDSC are the most potent suppressors among DC-HIL+myeloid cells induced by melanoma.

DC-HIL is Expressed by MDSC in Melanoma-Bearing Mice.

The inventors then examined expression of DC-HIL on MDSC purified frommice with melanoma (FIG. 3B). Purified MDSC were fractionated into theGr-1low monocytic (21%) and the Gr-1hgh granulocytic subsets (75%)(Movahedi et al., 2008 and Peranzoni et al., 2010). They comparedexpression of DC-HIL with other coinhibitory ligands expressed by MDSC(FIG. 3B). DC-HIL was expressed by 40% of MDSC (40-60% of eithersubset), PD-L1 by 26% (all of which were Gr-1low cells; rarely Gr-1highcells), and CD80 and CD86 by 66-73% regardless of subset. Importantly,MDSC isolated from tumor-free mice did not express DC-HIL at all (FIG.3B). More rigorously, they examined DC-HIL expression by MDSC subsets(FIG. 4C). CD11b+Gr-1+ MDSC sorted into 4 subsets based on differentialexpression of Ly6C vs. Gr-1 (Elkabets et al., 2010). The fractiondistinguished by Ly6ChighGr-1low phenotype (designated Fr. 1) expressedhighest levels of DC-HIL (37%); Ly6CmedGr-1high (Fr. 2) contained 11%DC-HIL+ cells; and the other 2 fractions (Ly6CmedGr-1low andLy6ClowGr-1low) expressed minimal DC-HIL just above background level.The inventors also determined the macrophage component of each fraction(FIG. 4E). Frs. 1 and 4 expressed low levels of F4/80 (14 and 18%,respectively); Fr. 2 was mixed with a small but distinct population ofmacrophages (23%); and Fr. 3 contained 45% macrophages.

Since MDSC phenotype within tissues may be heterogenous (Gabrilovich andNagaraj, 2009), the inventors examined whether DC-HIL is expressed bythese cells in tissues other than spleen. MDSCs isolated from BM,peripheral blood, and from the tumor site of melanoma-bearing mice, wereassayed for DC-HIL expression. DC-HIL was expressed by 30% of MDSC fromBM (FIG. 4A), 61% from blood (FIG. 4B), and 60% from the B16 tumor site(FIG. 4C). These results indicate that melanoma induces DC-HILexpression on MDSC in many organs, particularly of the Ly6ChighGr-1lowsubset.

DC-HIL Mediates the T Cell-Suppressive Function of MDSC.

The inventors then addressed whether MDSCs require DC-HIL expression tosuppress T cell activation. CD11b+Gr-1+ MDSC from melanoma-bearing mice(melanoma-MDSC) were incubated with pmel-1 spleen cells in the presenceof gp100 peptide at a 1:1 cell ratio. The inventors added UTX103anti-DC-HIL mAb (or control IgG) to block the function of DC-HILexpressed by MDSC (FIG. 5A). UTX103 mAb (but not control IgG) restoredpmel-1 T cell activation dose-dependently, with complete restoration atthe highest dose. Because APC in pmel-1 splenocytes express DC-HILalbeit at a lower level, they probed its influence by adding anti-DC-HILmAb to spleen cells with Ag but without MDSC. Neither UTX103 mAb norcontrol IgG affected T cell activation, indicating DC-HIL on spleen APChad no influence. The inventors also probed a role for othercoinhibitory ligands (FIG. 5B). Reproducibly, UTX103 mAb blockedsuppression completely, whereas anti-CD80 or anti-PD-L1 Ab did not;highest concentration of anti-CD86 Ab increased proliferation just 17%.Among coinhibitory ligands examined, DC-HIL was the only receptorcapable of mediating the suppressor activity of MDSC.

DC-HIL+ MDSC Subset is the Most Potent Suppressor Among the Total MDSCInduced by Melanoma.

Because only some (not all) MDSC induced by melanoma express DC-HIL(FIG. 3B) and because MDSC requires DC-HIL expression for their function(FIG. 5A), the inventors posited that DC-HIL+ MDSC are responsible forsuppressor activity. Because DC-HIL expression was confined to the Fr. 1subset, the inventors determined whether DC-HIL+ MDSC are more potentsuppressors than other subsets (Frs. 2-4). Unfractionated as well as the4 FACS-sorted fractions were added separately to culture of pmel-1spleen cells with gp100 peptide, and suppressive ability assayed (FIG.4E). Unfractionated MDSC inhibited T cell activation almost completely(100% suppression); Fr 1 (with highest DC-HIL expression level) alsoexhibited similarly strong activity; Fr. 2 exhibited weak activity evenat highest dose; and Frs 3 and 4 had no effect on T cells.

Because Fr. 1 was contaminated with DC-HIL^(neg) cells, the inventorsemployed a depletion strategy to examine whether DC-HIL+ MDSC are thepotent suppressors. DC-HIL+ cells were depleted from total CD11b+Gr-1+melanoma-MDSC and assayed for ability to suppress Ag-specific andnonspecific T cell activation. Reproducibly, untreated MDSC inhibitedgp100-triggered T cell activation almost completely, whereas depletionof DC-HIL+ cells abrogated the ability at every dose examined (FIG. 5C).

The inventors also questioned whether DC-HIL+ MDSC can induce CD8+ Tcell suppression in mice. On day 0, mice were injected with naive pmel-1CD8+ T cells, and on day 3 were given untreated or DC-HIL-depleted MDSCand vaccinated with gp100 peptide. Ten days later, draining LN cellswere prepared and IFN-γ-secreting cells counted (FIG. 5D). Mice infusedwith CD8+ T cells but without MDSC produced very high numbers of IFN-γ-Tcells (61.6±7.0 cells/2×10⁵ LN cells). This number was markedlydecreased by co-infusion of untreated MDSC (containing DC-HIL+ cells)(down to 13±2 cells), whereas suppression was abrogated by depletion ofDC-HIL+ cells from the MDSC preparation (60±5 cells). Altogether, theseresults indicate that the DC-HIL+ subset is responsible for suppressoractivity and for immunosuppression by CD11b+Gr-1+ MDSC inmelanoma-bearing mice.

Deletion of DC-HIL Abrogates MDSCs Ability to Suppress T Cell Activationand to Promote Melanoma Progression.

The inventors next studied the impact of DC-HIL gene deletion on MDSCfunction. MDSC isolated from DC-HIL KO or WT mice bearing melanoma wereassessed for their capacity to suppress activation of pmel-1 T cells.MDSC-depleted fraction (CD11b^(neg)) was also isolated from the samemice (control). Varying numbers of MDSC or CD11b^(neg) cells werecocultured with pmel-1 spleen cells plus Ag, and their proliferationmeasured (FIG. 5G). DC-HIL^(+/+) MDSC inhibited proliferationdose-dependently, with complete inhibition at 1:1 cell ratio. Bycontrast, DC-HIL^(−/−) MDSC failed to inhibit T cell activation, exceptfor minuscule effect at the highest ratio. CD11b^(neg) cells from WT orKO mice showed no inhibition. DC-HIL^(−/−) MDSC were confirmed to notexpress DC-HIL (FIG. 5G).

To assess the ability of MDSCs to promote melanoma growth, the inventorspurified melanoma-MDSC and co-injected them with B16 cells s.c. intonaive WT mice. A week later, melanoma-MDSC alone were infused i.v. intocorresponding mice (FIG. 5H). Melanoma in mice co-injected withDC-HIL^(+/+) MDSCs grew significantly larger than in mice injected withB16 cells alone (0.9±0.2 vs. 2.6±0.3 cm³ by day 17). By contrast,melanoma in mice co-injected with DC-HIL^(−/−) MDSCs grew similarly ascontrol (without MDSC). Loss of DC-HIL expression most likely led toreduced ability to suppress T cell activation and promote melanomagrowth.

To examine the possibility that deficient function of DC-HIL^(−/−) MDSCsresulted from reduced expression of soluble inhibitory mediators,melanoma-MDSC from WT or KO mice were compared for NO production,arginase activity, and ROS (FIGS. 6A-B). DC-HIL^(−/−) MDSCs expressed30% lower NO (13.3±1.6 vs. 19.1±2.3 μM) and 40% lower arginase activity(0.9±0.2 vs. 1.5±0.2 U/L) than WT control, whereas ROS was similar forboth. Thus, DC-HIL deficiency led to 30-40% reduction in constitutiveexpression of NO and arginase I. Because such impact is not sufficientto account for the much greater loss of suppressor activity byDC-HIL^(−/−) MDSC, the inventors do not think that constitutively lowlevels of soluble mediators accounted for loss of suppressor activity byDC-HIL−/− MDSC.

IFN-γ and NO Also Mediate the T Cell-Suppressive Activity of MDSCs.

Various soluble inhibitory mediators and cytokines were shown to mediatesuppression by MDSCs, but with no consistency among tumor models(Gabrilovich and Nagaraj, 2009). The inventors thus wanted to identifysoluble factors responsible for MDSC activity, independent of DC-HIL, byadding specific inhibitors to cocultures of pmel-1 spleen cells andmelanoma-MDSC. Neutralizing Ab to TGF-β (Filipazzi et al., 2007) orIL-10 (Wang et al., 2010) had no effect, whereas anti-IFN-γ Ab blockedsuppression completely (FIG. 7A). N-hydroxyl-nor-arginine (inhibitor ofarginase activity) or 1-methyl-tryptophan (indoleamine inhibitor)restored T cell activation marginally (FIG. 7B). Catalase and superoxidedismutase (ROS inhibitors) had little-to-no effect. By contrast,L-NG-monomethyl-arginine citrate (NOS inhibitor) orN6-(1-iminoethyl)-L-lysine (NOS-2 inhibitor) blocked MDSC function.Thus, IFN-γ and NOS-2 independently support the suppressor activity ofMDSC induced by B16 melanoma.

T Cell Expression of SD-4 is Required for MDSC Suppressor Activity.

Since ligation of SD-4 on activated T cells is required for DC-HIL onAPC to mediate inhibition (Chung et al., 2007), the inventors posited asimilar mechanism for MDSC. They tested the ability of melanoma-MDSC toinhibit activation of pmel-1 spleen cells in the presence of DC-HIL-Fc,which interferes with DC-HIL/SD-4 binding (Chung et al., 2007). LikeUTX103 mAb, DC-HIL-Fc restored T cell activation dose-dependently (up to90%), whereas control Ig did not (FIG. 7C). For SD-4 specificity, theinventors compared MDSCs ability to suppress activation of SD-4^(+/+)vs. SD-4^(−/−) pmel-1 spleen cells triggered by gp100 Ag (FIG. 7D).Without MDSC, spleen cells from both mice exhibited similar capacity toinduce T cell proliferation; whereas addition of MDSC to spleen cellsinhibited SD-4+/+ T cell activation dose-dependently (completely at 1:1cell ratio), while only 15% inhibition was achieved for SD-4^(−/−) Tcells at highest ratio. Thus, melanoma-MDSC are likely to inhibit T cellactivation via SD-4.

Crosslinking of DC-HIL Induces Tyrosine Phosphorylation Leading toExpression of IFN-γ and NOS-2 (iNOS) in MDSCs.

Since DC-HIL has an immunoreceptor tyrosine-based activation motif(ITAM) in its intracellular domain (Shikano et al., 2001), the inventorsposited that binding of DC-HIL to SD-4 not only inhibits T cellactivation via SD-4 signals but also activates MDSC function viaITAM-induced signals. They determined whether ligation of DC-HILtriggers tyrosine phosphorylation of the ITAM in MDSCs. DC-HIL onmelanoma-MDSC was cross-linked with UTX103 mAb, and expression oftyrosine-phosphorylation on DC-HIL examined (FIG. 7E). Tyrosinephosphorylation on DC-HIL was induced in MDSCs as early as 1 minfollowing cross-linking of the receptor, whereas control IgG inducedonly basal level. Amounts of DC-HIL protein were similar amongimmunoprecipitate samples. The tyrosine phosphorylation is likely tooccur on the ITAM, as shown previously using transfection experiments(Chung et al., 2009).

Having shown that DC-HIL, IFN-γ and NOS-2 are independent mediators forthe suppressor activity of MDSC, the inventors posited that all 3components participate in a cascade initiated by DC-HIL-linked tyrosinephosphorylation. The inventors thus examined expression of IFN-γ mRNA inMDSC after cross-linking DC-HIL receptor (FIG. 7F). IFN-γ mRNA wasmarkedly increased (20-fold greater than control) one day aftercross-linking of DC-HIL, and quickly decreased (but still 4-foldgreater) next day. To address specificity of cross-linking to IFN-γ, theinventors measured cytokine secretion by MDSC treated with UTX103 mAb(FIG. 7G). Among cytokines examined, IFN-γ was the most responsivecytokine, increasing 45-fold compared to control treatment on day 1after cross-linking. TNF-α secretion also increased, but only 4-5-foldhigher than control. Expression of IL-10 and TGF-3, which were notinvolved in suppressor activity (FIG. 7A), were unchanged. The inventorsthen examined expression of NOS-1, NOS-2, and NOS-3 mRNA (FIG. 7H).Cross-linking DC-HIL on MDSC induced NOS-2 expression 4-fold greaterthan control; it was the most abundantly expressed among the 3 types ofNOS, consistent with known NOS-2 inducibility by IFN-γ (Kamijo et al.,1994). Such high expression was supported by increased level of NO aftercross-linking DC-HIL on MDSCs (FIG. 7I).

Anti-DC-HIL Treatment of Melanoma-Bearing Mice Suppresses Tumor Growthand Reduces Circulating MDSC.

Since anti-DC-HIL mAb blocks MDSC suppressor function, the inventorsassessed effects of UTX103 mAb on melanoma growth. Because MDSC startedto accumulate in spleen 6 days after tumor inoculation (tumors grew to˜0.1 cm³) (Cheng et al., 2008), they injected UTX103 mAb or control IgGi.p. on this date and every other day after, for 6 treatments. In micetreated with control IgG, melanoma grew aggressively after day 10, inproportion to frequency of MDSCs in blood, which increased from 3% onday 0 to 32% on day 18. Treatment with UTX103 mAb markedly suppressedsubsequent growth of melanoma (FIG. 8A) and prevented MDSC fromexpanding in blood (FIG. 8B-C). Thus, anti-DC-HIL mAb treatmentsuppresses growth of established melanoma and blocks expansion of MDSCs.

Inhibited MDSC Function Accounts for the Therapeutic Effects ofAnti-DC-HIL mAb on Melanoma.

Having shown melanoma to express DC-HIL with inhibition of T cellactivation (Chung et al., 2007 and Tomihari et al., 2010), the inventorsprobed the basis for beneficial outcomes of UTX103 mAb. For DC-HIL+melanoma, they implanted WT mice with DC-HIL-knocked-down B16 cells(KD-B16) (Tomihari et al., 2010) instead of parental cells, with Abinjected starting day 11 and every other day after, for 5 treatments(FIG. 8D). In this assay, both MDSC and APC were DC-HIL+ cells. KD-B16tumor grew to 3.0 cm³ by day 21 in mice injected with control IgG, butits growth was inhibited markedly (1.0 cm³) in mice treated with UTX103mAb, negating the significant involvement of DC-HIL on melanoma to theeffects of anti-DC-HIL mAb.

For DC-HIL+ APC (FIG. 8E), BM-DC were pulsed with gp100 peptide andinfused into DC-HIL−/− mice bearing melanoma (−0.2 cm³), that were theninjected with CFSE-labeled pmel-1 T cells. On days 0 and 2, UTX103 mAbor control IgG was injected. Mice injected with unpulsed DC and pmel-1 Tcells served as control. On day 3, spleen or LN cells were harvested andexamined for CFSE fluorescence intensity of infused Thy1.1+ T cells. Inthis assay, both B16 cells and adoptively transferred DC were DC-HIL+cells. Proliferating pmel-1 T cells in spleen and LN of mice injectedwith Ag-pulsed DC/control IgG were significantly higher than in miceinjected with unpulsed DC. However, the proportion of proliferating Tcells was not significantly augmented by UTX103 mAb, indicating thatinfused anti-DC-HIL mAb had little-to-no effect on APC function of DC.

DC-HIL+ MDSC are Expanded in Blood of Melanoma Patients.

Since CD14⁺HLA-DR^(no/low) MDSC are the human equivalent of mouseCD11b⁺Gr-1⁺ MDSC (Filipazzi et al., 2007), we posited that bloodCD14⁺HLA-DR^(no/low) cells in melanoma patients express DC-HIL and thatsuch expression makes them immunosuppressive. Thus we examined bloodfrequencies of CD14⁺HLA-DR^(no/low) cells and their DC-HIL expression,in cases of: melanoma with varying clinical stages (0-IV) (n=62),dysplastic nevi (in which melanocytes are abnormal but not malignant(n=12)), and healthy donors (n=21) (FIG. 12A, FIG. 13). Compared tohealthy donors, all cases of melanoma exhibited elevated bloodCD14⁺HLA-DR^(no/low) cells (FIG. 12B), consistent with a prior report(Filipazzi et al., 2007). Whereas blood CD14⁺HLA-DR^(no/low) cells inhealthy donors had little-to-no expression of DC-HIL (0.1±0.1% DC-HIL⁺cells among PBMCs), all cases of metastatic melanoma (stages III/IV)displayed high-level DC-HIL expression on these cells (2.9±0.9% and2.6±0.6%, respectively; t test p=0.001 vs. healthy donors) (FIG. 12C).Intermediate levels of DC-HIL expression were seen in bloodCD14⁺HLA-DR^(no/low) cells of melanoma confined to skin (stages 0/I-II).Dysplastic nevi showed lower expression than skin-restricted melanoma,but higher than for healthy donors (p=0.01). Thus blood levels ofDC-HIL⁺CD14⁺HLA-DR^(no/low) cells correlated with cancer progression,particularly in advanced stages. Other MDSC subsets (CD14⁺IL-4Rα⁺,CD14^(neg)CD11b⁺CD15⁺, and CD14^(neg)IL-4Rα⁺CD15⁺) also expressed DC-HILat a range of 30-75% (FIG. 16).

To determine whether melanoma was the cause of the elevated bloodlevels, we followed a new cohort of 9 patients with stage 0 melanoma andassayed for % DC-HIL⁺CD14⁺HLA-DR^(no/low) MDSC in their PBMCs (FIG.12D), at 0, 1, 3, and 6 months after excision of the melanoma. At thetime of resection (0 month), all subjects except one (subject M83)exhibited higher levels than healthy controls (0.3 to 12.8%) (FIG. 14).Across the 3-month follow-up, these elevated levels declinedsignificantly in 8 patients (Wald test, p=0.045) to an average of 0.4%,close to that of 6 normal controls (FIG. 15). Interestingly, in the caseof one patient (M71), the % DC-HIL⁺CD14⁺HLA-DR^(no/low) cells thatdeclined a month post-resection climbed back to a high level at 3months, which coincided with discovery of a new melanoma in situ (stage0), and then fell back after resection of this second melanoma. Theinventors concluded that melanoma is responsible (directly orindirectly) for acquisition of DC-HIL expression by CD14⁺HLA-DR^(no/low)cells. Because our mouse studies showed IFN-γ and IL-1β to induce DC-HILexpression by CD11b⁺Gr1⁺ cells, we speculate similar mechanisms forhuman CD14⁺HLA-DR^(no/low) cells.

DC-HIL is a critical mediator of the T cell suppressor function of humanMDSC.

Do CD14⁺HLA-DR^(no/low) MDSC from melanoma patients suppress T-cellfunction and is DC-HIL responsible for that function?CD14⁺HLA-DR^(no/low) cells isolated from melanoma patients (vs. healthydonors) were cocultured with autologous T-cells activated byanti-CD2/CD3/CD28 Ab (FIG. 17A). CD14⁺HLA-DR^(no/low) cells frommelanoma patients inhibited IFN-γ production by autologous T-cellsdose-dependently and almost completely, whereas corresponding cells fromhealthy donors were weakly immunosuppressive.

Treatment with anti-DC-HIL mAb (but not control IgG) restored the T-cellIFN-γ response dose-dependently (up to 80%) (FIG. 17B, FIG. 18).Moreover, treatment of total (unfractionated) PBMCs from melanomapatients with anti-DC-HIL mAb (but not with control IgG) enhanced theIFN-γ response, and this enhancement correlated positively with melanomastaging (FIG. 17C), but negatively with IFN-γ levels from IgG-treatedPBMCs (FIG. 17D).

DC-HIL⁺ MDSC are Also Expanded in Blood of Non-Melanoma Cancer Patients.

DC-HIL expression on MDSC was also examined in blood of patients (stagesIII or IV) with a cancer form of cutaneous squamous cells, breast,colon, or lung (FIG. 19). DC-HIL expression is indicated as % DC-HIL⁺MDSC among total PBMCs. Almost all patients (n=6-10) with squamous cellcarcinoma, breast, and colon cancers induced high expression of DC-HIL,compared to healthy donors. Expression was very low in lung cancers.Thus, expansion of blood DC-HIL⁺ MDSC is not confined to melanoma.

Identification of Complementary Determining Regions (CDRs) in MouseAnti-Human DC-HIL mAb Clones.

Immunization of BALB/c mice with DC-HIL-Fc recombinant protein(consisting of the extracellular domain of human DC-HIL fused to theIgG-Fc) led to the generation of 2 different mAb clones (3D5 and 4G5)with high affinity and specificity to DC-HIL protein in Western blottingand flow-cytometry (FIG. 11A-C). These clones consist of an IgG1 heavychain and a κ light-chain. For determining DNA sequences of the mAb,total RNA isolated from the mAb clones was PCR-amplified with primersencoding for the signal peptides and for the amino acid sequences of theCγ1 region (FIGS. 9A-B and 10A-B). The resulting PCR products wereDNA-sequenced and, the amino acid sequences were deduced. Using acomputer-analysis, CDRs are determined. Only one amino acid in the3^(rd) CDR of the Vκ is disparate between 3D5 (FIG. 9B) and 4G5 (FIG.10B).

High Expression of DC-HIL on MDSC in the Blood of Patients with CancersOther than Melanoma.

The inventors questioned whether cancers other than melanoma displayhigh expression of DC-HIL on MDSC in the blood of patients. For thisstudy, we recruited new cases of cancer patients with breast, colon,kidney, lung, pancreatic, and non-melanoma skin cancers (basal cellcarcinoma (BCC) and cutaneous squamous cell carcinoma (SCC)). Allpatients were metastatic (stages III or IV) and untreated. Blood sampleswere taken and analyzed by flow-cytometry for frequency (%) of DC-HIL⁺MDSC among total peripheral blood monocytes (PBMC), using 3D5anti-DC-HIL mAb.

The inventors' data showed that metastatic melanoma patients display2.5% (median value) of DC-HIL⁺ MDSC among total PBMCs, with the rage of0.8-4.4% (FIG. 4C).

Healthy controls showed 0.05%, 0.01-0.05%, n=21 (FIG. 4C). In thepresent studies, cancer patients who showed higher levels than melanomaare: colon cancer (median of 3.2% and the range of 0.1-27%, n=32); andkidney cancer (3.3%, 1.0-4.8%, n=5) (FIG. 25). Cancers with lower levelsthan melanoma but significantly higher than healthy controls are: BCC,1.22%, 0.02-7.41%, n=29; SCC, 1.4%, 0.02-5.4%, n=25; breast cancer(1.3%, 0.5-5.5%, n=10); and pancreatic cancer (1.6%, 0.01-17%, n=10). Acancer with very low levels, but slightly higher than healthy controls,is lung cancer (0.3%, 0-5.4%, n=20). Thus, high expression of DC-HIL onMDSC in the blood is noted in patients with almost all common cancertypes, except lung cancers. These results also indicate the possibilitythat many cancers can be targets for treatment of humanized anti-DC-HILmAb.

Ability of 3D5 Anti-DC-HIL mAb to Neutralize the T Cell-SuppressorFunction of MDSC in Varying Cancers.

Previously we showed that 3D5 anti-DC-HIL mAb treatment neutralizealmost completely the T cell-suppressor function of MDSC isolated frommelanoma patients, using cell culture systems. To examine whether thesame mAb can similarly neutralize the function of MDSC from patientswith other cancer types, MDSC and T cells were purified from the bloodof the same patient and co-cultured in the presence of Tcell-stimulators (magnetic beads coated with anti-CD2/CD3/CD28 Ab), with3D5 mAb or control IgG (see the legends of FIGS. 21A-B). T cellactivation was measured by IFN-γ production. Effect of 3D5 mAb wasexpressed as % restoration of T cell IFN-γ response that was suppressedby MDSC. The inventors performed the assays using blood samples frompancreatic cancer (n=4), colon cancer (n=6), kidney (n=2), BCC (n=2),and SCC (n=2). The effect of 3D5 mAb was variable, depending on cancertypes and also on individuals. 3D5 mAb produced 80-100% restoration inall cases of pancreatic cancers (FIGS. 21A-B); 70-100% restoration forall cases of colon cancers (FIG. 22); just 25% for kidney cancers (Note:3D5 mAb treatment increased T cell response by 20-fold greater thancontrol IgG-treated cultures) (FIGS. 23A-B); ˜20-90% for BCC; and almostno effect for SCC (FIGS. 24A-B). Note no effects of control IgG on theMDSC function. These data indicate that 3D5 anti-DC-HIL mAb can reversethe T cell-suppressor function of MDSC from not only melanoma but alsofrom other cancers (colon, pancreatic, and kidney cancers). Thus,humanized anti-DC-HIL mAb are likely to produce beneficial outcomes incancer patients with high expression of DC-HIL on MDSC.

Example 3—Discussion

While the important detrimental effect of an expanding population ofcirculating MDSC in patients with growing cancers is well established,the mechanism underlying the profound immunosuppression induced by MDSCremain unclear. Like tolerogenic APC, soluble inhibitory mediators andcoinhibitory receptors were both reported to mediate MDSCs suppressorfunction, but no data has bridged these 2 mechanisms. The inventors nowprovide such linkage through the DC-HIL receptor that can trigger theIFN-γ/NOS-2 axis. Since DC-HIL expression confers the greatestsuppression to CD11b+Gr-1+ MDSC generated by melanoma, DC-HIL may beconsidered an activation marker for MDSC, akin to CD80/CD86costimulatory receptors for immune-stimulatory APC, but with acompletely polar effect.

Because CD80, CD86 (as ligands of CTLA-4), and PD-L1 are coinhibitoryligands that deliver negative signals to T cells through theircorresponding receptors (Egen et al., 2002 and Keir et al., 2011), theinventors tested their potential as a mediator of MDSCs suppressoractivity (Yang et al., 2006; Liu et al., 2008 and Liu et al., 2009).Using a melanoma mouse model, they showed MDSC to express CD80, CD86,and PD-L1 at levels similar or greater than DC-HIL. However, only theDC-HIL pathway was shown responsible for the T cell suppressor activityand the tumor-promoting effects of MDSC. This distinction was due to adiscovered bidirectional signal, in which DC-HIL on the one handactivates SD-4-linked protein tyrosine phosphatase (CD148) in T cells(Chung et al., 2011), while on the other hand also transduces tyrosinephosphorylation of its ITAM-like motif (only one unit of ITAM) (Humphreyet al., 2005), leading to activation of the IFN-γ/NOS-2 cascade in MDSC.These results suggest cross-linking of DC-HIL on MDSC induces activationof Syk kinase (Lowell et al., 2011), which is a major signal mediatorfor ITAM that in turn leads us to theorize a requirement forpreactivation of MDSC before it can exert its remarkable Tcell-suppressive activity. By contrast, other ligands that lackauthentic tyrosine-based signal motifs only induce a unidirectionalsignal via their T cell receptors (CTLA-4 and PD-1) (Carreno andCollins, 2002 and Sharpe et al., 2007).

One consequence of ligating DC-HIL/SD-4 on MDSC is secretion of IFN-γcritical to activation and T cell-suppressive activity of these cells,consistent with previous studies of MDSC isolated from mice with liverinflammation (Cripps et al., 2010) or with C26 colon carcinoma (Gallinaet al., 2006). In the latter study, MDSC suppressor activity requiredIFN-γ expression by both MDSC and T cells: MDSC from IFN-γ-deficientmice lost suppressor activity, and MDSC from WT mice failed to exertsuch activity on T cells lacking the IFN-γ gene. The former findingsupports the inventors' theory, whereas the latter outcome raises thepossibility that T cell-derived IFN-γ regulates sensitivity of T cellsto MDSC function. The inventors speculate that MDSC-secreted IFN-γ isinternalized rapidly by MDSC (prior to any T cell effect) and activatesits signal pathway by binding to nuclear receptors (intracellularactivation) (Puddu et al., 2005). Indeed, density of IFN-γ nuclearreceptors was reported greater than cell-surface receptors in some cells(MacDonald et al., 1986). On the other hand, T cell-derived IFN-γ mayaugment or maintain activated MDSC or sustain a high level of SD-4expression by T cells.

Because DC-HIL in this model was expressed by at least 3 cell types(melanoma, APC, and MDSC), their each compartment could have influencedthe outcomes. Thus, it was important to assess their respectivecontributions. Since the ability of anti-DC-HIL mAb to suppress melanomagrowth was not diminished in mice bearing melanoma knocked-down forDC-HIL and since the same mAb did not enhance the APC capacity of DCinjected into DC-HIL^(−/−) mice, the inventors conclude that DC-HIL+MDSC (and not DC-HIL+ melanoma nor DC-HIL+ APC) exerted the criticaleffect. This conclusion was also supported by in vitro assays showingdeletion of DC-HIL to abrogate MDSC suppressor function completely,while only increasing melanoma-induced activation of T cells by 50%(Tomihari et al., 2010) and only augmenting APC capacity 2-fold. Alsoconsistent with this conclusion is over-representation of DC-HIL+ MDSCwithin spleens of melanoma-bearing mice, contrasted with their very lowfrequencies in tumor-free mice. This is not to say DC-HIL+ melanomacells or DC-HIL+ APC play no roles in tumor development, since bothlikely contribute importantly to initial phases of primary melanomagrowth. However, the inventors believe that their effects becomesuperseded by the progressively greater influence of exponentiallyexpanding DC-HIL+ MDSC induced by the enlarging tumor.

Therapeutic agents targeting MDSC have been developed, including:all-trans retinoic acid (Lathers et al., 2004), which promotes MDSCdifferentiation to APC; the tyrosine kinase inhibitor sunitinib (Ko etal., 2009) that inhibits their metabolic process; COX-2 inhibitors(Rodriguez et al., 2005), Stat3 inhibitors (Xin 2009), and thephosphodiesterase-5 inhibitor (Sildanefil) (Serafini et al., 2006) whichblock or neutralize suppressor function. These agents improved efficacyof anti-tumor immunotherapy, but unlike the inventors' anti-DC-HIL mAb,none were specific for activated MDSC.

Humanized anti-CTLA-4 mAb (ipilimumab) is a recently developed treatmentfor metastatic melanoma that prolonged survival of patients withmetastatic melanoma (Hodi et al., 2010). Ipilimumab augments T celleffects by blocking the coinhibitory function of CTLA-4. However, itsbenefits are limited by development of autoimmune disease causingdermatitis, hepatitis, colitis, and in some cases, death (51). UnlikeDC-HIL, the CTLA-4 ligands CD80 and CD86 are not involved in MDSCfunction. CTLA-4 is present in all activated T cells and regulatesdevelopment of autoreactive T cells via supporting T_(reg) function(Gattinoni et al., 2006), whereas SD-4 is only expressed by effector Tcells (Akiyoshi et al., 2010) and has no impact on T_(reg) (unpublisheddata). Moreover, CTLA-4-mice die early from lymphoproliferative disease(Tivol et al., 1995), while DC-HIL^(−/−) or SD-4^(−/−) mice survivewithout observable autoimmune diseases (unpublished data). Thesedifferences suggest that strategies negating DC-HIL/SD-4 function mayyield safer or better outcomes than those directed at CTLA-4.

In summary, blocking DC-HIL function inhibited growth of establishedmelanoma and prevented MDSC expansion in mice, while restoringMDSC-suppressed T cell function in melanoma patients. These outcomesprovide a strong incentive for developing treatment modalities thattarget DC-HIL-dependent pathway.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this disclosure havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the disclosure. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the disclosure as defined by theappended claims.

VII. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-20. (canceled)
 21. A recombinant monoclonal antibody, or fragmentthereof, characterized by CDR sequences as follows: CDR1- CDR2- CDR3-CDR1- CDR2- CDR3- CLONE L L L H H H 3D5 SSISY TTS HQSSS GYTF INTR TTGF(SEQ (SEQ YPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ (SEQ NO: NO: ID ID IDID 1) 2) NO: NO: NO: NO: 3) 4) 5) 6) 4G5 SSISY TTS HQSSS GYTF INTR TTGF(SEQ (SEQ HPYT TNYY NGGN AY ID ID (SEQ (SEQ (SEQ (SEQ NO: NO: ID ID IDID 1) 2) NO: NO: NO: NO: 7) 4) 5) 6)


22. The recombinant monoclonal antibody or fragment of claim 21, whereinsaid antibody or antibody fragment is encoded by a light chain variablesequence according to SEQ ID NOS: 8 or 10 a sequence having 70%, 80%, or90% identity to SEQ ID NOS: 8 or 10, and a heavy variable chain sequenceaccording to SEQ ID NOS: 9 or 11 or a sequence having 70%, 80% or 90%identity to SEQ ID NOS: 9 or 11, respectively.
 23. The recombinantmonoclonal antibody or fragment of claim 21, wherein said antibody orantibody fragment is encoded by a light chain variable sequenceaccording to SEQ ID NOS: 8 or 10 a sequence having 95% identity to SEQID NOS: 8 or 10, and a heavy variable chain sequence according to SEQ IDNOS: 9 or 11 or a sequence having 95% identity to SEQ ID NOS: 8 or 10,respectively.
 24. The recombinant monoclonal antibody or fragment ofclaim 21, comprising light and heavy chain variable region sequencescomprising SEQ ID NO: 12 and SEQ ID NO:
 13. 25. The recombinantmonoclonal antibody or fragment of claim 21, comprising light and heavychain variable region sequences comprising SEQ ID NO: 14 and 15,respectively.
 26. The recombinant monoclonal antibody or fragment ofclaim 21, wherein said antibody is a single chain antibody.
 27. Therecombinant monoclonal antibody or fragment of claim 21, wherein saidantibody is a single domain antibody.
 28. The recombinant monoclonalantibody or fragment of claim 21, wherein said antibody is a chimericantibody.
 29. The recombinant monoclonal antibody or fragment of claim21, wherein said antibody a Fab fragment.
 30. The recombinant monoclonalantibody or fragment of claim 21, wherein said antibody is a recombinantantibody having specificity for DC-HIL and a second MDSC surfaceantigen.
 31. The recombinant monoclonal antibody or fragment of claim21, wherein said antibody is a murine antibody.
 32. The recombinantmonoclonal antibody or fragment of claim 21, wherein said murineantibody is an IgG.
 33. The recombinant monoclonal antibody or fragmentof claim 21, wherein antibody is a humanized antibody.
 34. Therecombinant monoclonal antibody or fragment of claim 21, wherein saidhumanized antibody is an IgG.
 35. A method of treating cancer comprisingadministering to said subject an antibody that binds to DC-HIL on thesurface of a myeloid-derived suppressor cell (MDSC).
 36. (canceled) 37.The method of claim 35, wherein said cancer is melanoma, lung cancer,brain cancer, head & neck cancer, breast cancer, skin cancer, livercancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer,uterine cancer, cervical cancer, ovarian cancer, testicular cancer, oresophageal cancer.
 38. The method of claim 35, further comprisingassessing the number of DC-HIL-positive MDSCs in said subject.
 39. Themethod of claim 38, further comprising assessing the level of DC-HIL onMDSCs from said subject.
 40. The method of claim 35, further comprisingtreating said subject with a second anti-cancer agent or treatment.41-65. (canceled)
 66. An antibody-producing cell expressing ananti-DC-HIL monoclonal antibody, wherein the antibody is characterizedby CDR sequences as follows: CDR1- CDR2- CDR3- CDR1- CDR2- CDR3- CLONE LL L H H H 3D5 SSISY TTS HQSSS GYTF INTR TTGF (SEQ (SEQ YPYT TNYY NGGN AYID ID (SEQ (SEQ (SEQ (SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 3)4) 5) 6) 4G5 SSISY TTS HQSSS GYTF INTR TTGF (SEQ (SEQ HPYT TNYY NGGN AYID ID (SEQ (SEQ (SEQ (SEQ NO: NO: ID ID ID ID 1) 2) NO: NO: NO: NO: 7)4) 5) 6)

67-79. (canceled)